Method for selecting a single cell expressing a heterogeneous combination of antibodies

ABSTRACT

Described are combinations of specific binding proteins, such as immunoglobulins, that are designed to be true combinations, essentially all components of the combination being functional and compatible with each other. Further provided are methods for producing a composition comprising at least two different proteinaceous molecules comprising paired variable regions, the at least two proteinaceous molecules having different binding specificities, comprising paired variable regions, at least two proteinaceous molecules having different binding specificities, comprising contacting at least three different variable regions under conditions allowing for pairing of variable regions and harvesting essentially all proteinaceous molecules having binding specificities resulting from the pairing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/140,321, filed Apr. 27, 2016, pending, which is acontinuation-in-part of co-pending U.S. patent application Ser. No.12/931,955, filed Feb. 14, 2011, which is a continuation of U.S. patentapplication Ser. No. 11/292,414, filed Nov. 30, 2005, now U.S. Pat. No.7,919,257, which is a continuation of PCT International PatentApplication No. PCT/NL2004/000386, filed on May 28, 2004, designatingthe United States of America, and published in English, as PCTInternational Publication No. WO 2004/106375 A1 on Dec. 9, 2004, whichapplication claims priority to European Patent Application No.03076671.1 filed on May 30, 2003, the contents of the entirety of eachof which is incorporated herein by this reference.

This application is also a continuation-in-part of co-pending U.S.patent application Ser. No. 12/589,181, filed Oct. 19, 2009, pending,which application is a continuation of U.S. patent application Ser. No.12/459,285, filed Jun. 29, 2009, now abandoned, which applications claimthe benefit, under 35 U.S.C. §119(e), to U.S. Provisional PatentApplication Ser. No. 61/133,274, filed Jun. 27, 2008, the entirecontents of each of which are hereby incorporated herein by thisreference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)-SEQUENCE LISTINGSUBMITTED AS A TXT FILE

Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The application relates to the field of molecular biology, inparticular, to medical molecular biology.

BACKGROUND

Specific recognition plays an important role in modern medical biology.Receptor-ligand interactions, immune responses, infections, enzymaticconversions are all based on specific recognition between molecules. Ofparticular interest are specific protein-protein interactions, whichgive a vast array of possibilities to interfere in all kinds ofbiological processes. Throughout nature, biological processes are foundthat depend on more than one (simultaneous) protein-interaction. At thepresent time, it seems that interfering at more than one point in abiological process is going to be more effective than a singleinterference. Particularly in antibody therapy, it is seen that one(monoclonal) antibody is often not effective enough for treating aparticular disorder and/or disease. Therefore, the attention of manymedical researchers is now focused on combination therapies. Well-knownexamples of combinations of antibodies that are presently clinicallypursued are for the treatment of non-Hodgkin's lymphoma, the combinationof the already approved anti-CD20 antibody Rituxan with the anti-CD22antibody Epratuzumab from AmGen, and for the treatment of Hepatitis B, acombination of two human antibodies being developed by XTLPharmaceuticals (E. Galun et al., Hepatology (2002) 35:673-679).However, the combination of multiple (two or more) drugs (be itantibodies or other) has a number of technical, practical and regulatorydrawbacks. The drugs were typically not designed as combinations anddevelopment with optimal clinical efficacy and compatibility may be aproblem. As an example, conditions for stabilizing the one may bedetrimental to stability of the other(s). Furthermore, multiple sourcesof recombinant production lead to multiple sources of risks, such as,viral contamination, prion contamination and the like.

B cells mediate humoral immunity by producing specific antibodies. Thebasic structural subunit of an antibody (Ab) is an immunoglobulin (Ig)molecule. Ig molecules consist of a complex of two identical heavy (H)and two identical light (L) polypeptide chains At the amino terminus ofeach H chain and L chain is a region that varies in amino acid sequencenamed the variable (V) region. The remaining portion of the H and Lchains is relatively constant in amino acid sequence and is named theconstant (C) region. In an Ig molecule, the H and L chain V regions (VHand VL) are juxtaposed to form the potential antigen-binding site. Thegenes that encode H and L chain V regions are assembled somatically fromsegments of germline DNA during precursor B (pre-B) celldifferentiation: V, D and J gene segments for the H chain and V and Jgene segments for the L chain. Within Ig V regions are three regions ofgreatest amino acid sequence variability that interact to form theantigen-recognition site and are thus referred to as complementaritydetermining regions (CDRs).

The V gene segment encodes the bulk of the V region domain, includingCDR1 and CDR2. Diversity in CDR1 and CDR2 derives from sequenceheterogeneity among multiple different germline-encoded V segments. CDR3is encoded by sequences that are formed by the joining of H chain V, D,and J gene segments and L chain V and J segments and by mechanisms thatcreate nucleotide sequence heterogeneity where these segments arecombined. Additional diversity may be derived from pairing of differentH and L chain V regions. Collectively these processes yield a primaryrepertoire of antibodies encoded by germline gene segments and expressedby newly formed B cells.

An additional source of antibody diversity is imposed on top of thediversity generated by recombination of Ig gene segments. B cells areable to introduce mutations into the antibody V regions that theyexpress, a process called somatic hypermutation. Thus, when an animalfirst encounters an antigen, the antigen binds to a specific B cellwhich happens to carry antibodies which have a V domain which binds theantigen. This primary response may activate this B cell to go on tosecrete the cognate antibody. These activated B cells can also nowtarget a somatic mutation process to their rearranged antibody genesegments and thus allow the production of daughter cells which makevariants of the antibodies of the primary response. A selection processamplifies those variant B cell descendants which make an antibody ofimproved affinity of the antigen. In B cells, somatic hypermutations aretargeted to a restricted genomic region including both the rearranged VHand VL genes. Thus somatic mutation allows affinity maturation—theproduction and selection of high affinity antibodies. Therefore, somaticmutation is important for the generation of high affinity antibodies.

The exquisite specificity and high affinity of antibodies and thediscovery of hybridoma technology allowing the generation of monoclonalantibodies (mAbs) has generated great expectations for their utilizationas targeted therapeutics for human diseases. MAbs are identical becausethey are produced by a single B cell and its progeny. MAbs are made byfusing the spleen cells from a mouse that has been immunized with thedesired antigen with myeloma cells to generate immortalized hybridomas.One of the major impediments facing the development of in vivoapplications for mAbs in humans is the intrinsic immunogenicity ofnon-human Igs. Patients respond to therapeutic doses of mouse mAbs bymaking antibodies against the mouse Ig sequences (Human Anti MouseAntibodies; HAMA), causing acute toxicity, alter their biodistributionand accelerate clearance, thus reducing the efficacy of subsequentadministrations (Mirick et al. (2004), Q. Nucl. Med. Mol. Imaging48:251-257).

To circumvent the generation of HAMA, antibody humanization methods havebeen developed in an attempt to produce mAbs with decreasedimmunogenicity when applied to humans. These endeavors have yieldedvarious recombinant DNA-based approaches aimed at increasing the contentof human amino acid sequences in mAbs while retaining the specificityand affinity of the parental non-human antibody. Humanization began withthe construction of mouse-human chimeric mAbs (S. L. Morrison et al.(1984), Proc. Natl. Acad. Sci. USA 81:6851-5), in which the Ig C regionsin murine mAbs were replaced by human C regions. Chimeric mAbs contain60-70% of human amino acid sequences and are considerably lessimmunogenic than their murine counterparts when injected into humans,albeit that a human anti-chimeric antibody response was still observed(W. Y. Hwang et al. (2005), Methods 36:3-10).

In attempts to further humanize murine mAbs, CDR grafting was developed.In CDR grafting, murine antibodies are humanized by grafting their CDRsonto the VL and VH frameworks of human Ig molecules, while retainingthose murine framework residues deemed essential for specificity andaffinity (P. T. Jones et al. (1986), Nature 321:522). Overall,CDR-grafted antibodies consist of more than 80% human amino acidsequences (C. Queen et al. (1989), Proc. Natl. Acad. Sci. U.S.A.86:10029; P. Carter et al. (1992), Proc. Natl. Acad. Sci. U.S.A.89:4285). Despite these efforts, CDR-grafted, humanized antibodies wereshown to still evoke an antibody response against the grafted V region(W. Y. Hwang et al. (2005), Methods 36:3).

Subsequently to CDR grafting, humanization methods based on differentparadigms such as resurfacing (E. A. Padlan et al. (1991), Mol. Immunol.28:489), superhumanization (P. Tan D. A. et al. (2002), J. Immunol.169:1119), human string content optimization (G. A. Lazar et al. (2007),Mol. Immunol. 44:1986) and humaneering have been developed in an attemptto further decrease the content of non-human sequences in therapeuticmAbs (J. C. Almagro et al. (2008), Frontiers in Bioscience 13:1619). Asin CDR grafting approaches, these methods rely on analyses of theantibody structure and sequence comparison of the non-human and humanmAbs in order to evaluate the impact of the humanization process intoimmunogenicity of the final product. When comparing the immunogenicityof chimeric and humanized antibodies, humanization of variable regionsappears to decrease immunogenicity further (W. Y. Hwang et al. (2005),Methods 36:3-10).

De-immunization is another approach developed to reduce theimmunogenicity of chimeric or mouse antibodies. It involves theidentification of linear T-cell epitopes in the antibody of interest,using bioinformatics, and their subsequent replacement by site-directedmutagenesis to human or non-immunogenic sequences (WO 09852976A1, thecontents of which are incorporated by this reference). Althoughde-immunized antibodies exhibited reduced immunogenicity in primates,compared with their chimeric counterparts, some loss of binding affinitywas observed (M. Jain et al. (2007), Trends in Biotechnol. 25:307).

The development of phage display technology complemented and extendedhumanization approaches in attempts to obtain less immunogenic mAbs fortherapy in humans. In phage display, large collections (“libraries”) ofhuman antibody VH and VL regions are expressed on the surface offilamentous bacteriophage particles. From these libraries, rare phagesare selected through binding interaction with antigen; soluble antibodyfragments are expressed from infected bacteria and the affinity ofbinding of selected antibodies is improved by mutation (G. Winter et al.(1994), Annu. Rev. Immunol. 12:433). The process mimics immuneselection, and antibodies with many different bindings specificitieshave been isolated using this approach (H. R. Hoogenboom et al. (2005),Nat. Biotechnol. 23:1105). Various sources of H and L chain V regionshave been used to construct phage display libraries including thoseisolated from non-immune or immune donors. In addition, phage displaylibraries have been constructed of V regions that contain artificiallyrandomized synthetic CDR regions in order to create additionaldiversity. Often, antibodies obtained from phage display libraries aresubjected to in vitro affinity maturation to obtain high affinityantibodies (H. R. Hoogenboom et al. (2005), Nat. Biotechnol. 23:1105).

The creation of transgenic mouse strains producing human antibodies inthe absence of mouse antibodies has provided another technology platformfor the generation of specific and high affinity human mAbs forapplication in humans. In these transgenic animals, the endogenous mouseantibody machinery is inactivated and replaced by human Ig loci tosubstantially reproduce the human humoral immune system in mice (A.Jakobovits et al. (2007), Nat. Biotechnol. 25:1134; N. Lonberg (2005),Nat. Biotechnol. 23:1117). B cell development as well as Igdiversification by recombination of gene segments is faithfullyreproduced in these mice, leading to a diverse repertoire of murine Bcells expressing human Igs. By immunizing these mice with antigens, itwas further demonstrated that these transgenic animals accumulatedsomatic mutations in the V regions of both heavy and light chains toproduce a wide diversity of high-affinity human mAbs (N. Lonberg (2005),Nat. Biotechnol. 23:1117).

The question, whether “fully human” mAbs such as derived from phagedisplay libraries or transgenic mice are less immunogenic than humanizedmAbs cannot be answered yet, because full immunogenicity data areavailable for just two human mAbs. An anti-tumor necrosis factor mAb,developed from phage-displayed human libraries induced antibodyresponses in 12% of patients—at the higher end of the incidence ofanti-antibody responses of the humanized antibodies (W. Y. Hwang et al.(2005), Methods 36:3-10).

Evaluation of the immunogenicity of the first registered human mAbgenerated by the transgenic approach demonstrated that mAb treatmentresulted in the generation of antibodies in approximately 5.5% oftreated cancer patients (A. Jakobovits et al. (2007), Nat. Biotechnol.25:1134; J. A. Lofgren et al. (2007), J. Immunol. 178:7467)

SUMMARY OF THE INVENTION

Described are combinations of specific binding proteins, such asimmunoglobulins, that are designed to be true combinations, essentiallyall components of the combination being functional and compatible witheach other. By producing true combinations, an avenue of furtherimprovements in both the production and properties of the combinationshas been opened up.

Disclosed are methods for producing a composition comprising at leasttwo different proteinaceous molecules comprising paired variableregions, the at least two proteinaceous molecules having differentbinding specificities, comprising contacting at least three differentvariable regions under conditions allowing for pairing of variableregions and harvesting essentially all proteinaceous molecules havingbinding specificities resulting from the pairing. Binding specificitiesare defined as interactions between molecules that can be distinguishedfrom background interactions. Typically, specific interactions betweenmolecules have higher binding affinity than background interactionsbetween molecules.

Specific binding molecules, which for an important part are made up ofamino acid residues (proteinaceous molecules), often require the pairingof different amino acid sequences in order to build a binding site. Anamino acid sequence that pairs with another amino acid sequence to builda binding site is referred to as a “variable region” herein. Of course,such a sequence may be part of a larger amino acid sequence, which mayagain be part of a larger proteinaceous molecule, e.g., as a subunit. Asan example, in an antibody a complementarity-determining region (CDR)may be a variable region, but a combination of three CDRs with theirframework regions may also be considered as a variable region. Asdecribed herein, at least two different binding sites may be built inone system, in one method. Thus, variable regions (amino acid sequencesor “peptides”) are brought together under conditions in which they maypair to build two different binding sites. This requires at least threevariable regions, of which one is capable of pairing with both othervariable regions, thus building two specific binding sites. The twospecific binding sites may be in one proteinaceous molecule or indifferent proteinaceous molecules, or both.

In antibodies of the IgG isotype, for example, this would be an antibodyhaving two identical or two different binding sites. By producing thetwo desired binding specificities in one system, there is only onesource of the products and thereby less risk of contamination withviruses, prions and the like. Such a system may be a cell-free system,such as a wheat germ system, but the described methods may be carriedout inside a cell, or more cells of the same origin, or of the origin ofthe subjects to be treated, typically human For production and selectionpurposes, other cells, such as, bacteria, insect cells, yeasts and othereukaryotes may be used.

If the pairing of the variable regions takes place in a cell, then theproduction of the variable regions may also take place in a cell,including the same cell. A particularly useful way of producing variableregions is through the expression of nucleic acids encoding thesevariable regions. In certain embodiments, all variable regions in onecell are produced by such expression, it is, however, also possible toproduce a number of variable regions in this manner and have othervariable regions brought in, based on different techniques ofproduction, or the same means of production, but in another cell. Formost purposes, the nature of the nucleic acid is not critical, it may beRNA or DNA, may be episomal or integrated, part of a viral vector or aplasmid, etc. However, for the final production system of thecombination of proteins having different binding specificities, thenucleic acid or acids encoding the variable regions may be stablyintegrated into the host genome. Production of variable regions throughexpression of nucleic acids encoding them gives the possibility tomanipulate the encoding sequences, thereby enabling the designing of newbinding specificities, better pairing properties, exchanging usefulsequences from one encoding sequence to another and the like. It alsogives the possibility for selection for improved or different bindingand/or pairing properties after alterations have been made, giving riseto the creation of libraries of many different nucleic acids in systemswith easy selection mechanisms.

In this manner, the number of variable regions to be expressed forobtaining different binding sites may be reduced. One may design and/orselect for a so-called “promiscuous” variable region, which is capableof pairing with more than one different binding region. “Pairing” isdefined herein as any kind of coming together to build a binding site,be it through covalent or noncovalent bonding, conformationalarrangement, folding, dimerization, multimerization or any other way. Itthus encompasses terms such as associating, assembling, binding,combining and the like, be it directly or indirectly. Particularly whenmore than two different binding specificities are made in one cell, itis useful to have promiscuous variable regions in such a system,reducing the number of different nucleic acids that have to beexpressed. In such a system, the promiscuous variable region should notcontribute significantly to the binding specificity of the pairedregions. In embodiments, the promiscuous variable region it is mostlyinvolved in folding and stability of the binding site, thereby, ofcourse, indirectly influencing the binding specificity.

Apart from reducing the number of nucleic acids to be expressed, bychoosing one or more promiscuous variable regions, the number of pairedvariable regions which are not functional can be reduced to essentiallyzero.

Particularly in the field of immunoglobulins, which typically comprisetwo pairs of two different paired variable regions, the production ofmore than one immunoglobulin inside the same cell often leads to pairingof variable regions that does not lead to a desired binding specificity.As decribed herein, pairs may be designed such that in one systemessentially all variable regions can pair with another in the system toform a useful specific binding site. In methods of the prior art whereinfour variable regions were expressed in hybrid-hybridomas or quadromas,the result was a low percentage of desired bispecific antibodies, apercentage of either original antibodies and a substantial percentage ofpaired regions without significant useful binding specificity.Bispecific antibodies may be produced with the described methods, eithertogether with or without the concomitant production of the originalantibodies, but typically essentially without production ofnon-functional pairs. In addition, mixtures of multiple monoclonal andmultiple bispecific antibodies may be produced with the methods.

The methods as disclosed in the detailed description provide foradaptation of the nucleic acids encoding variable regions to the desiredend result. Using promiscuous pairing or the opposite, monogamouspairing, the end result can be designed. Where bispecific antibodies orother certain pairings are to be excluded, the use of pairs of variableregions that can pair only with each other is used. Further, methods asdisclosed in the detailed description provide for adaptation of thenucleic acids encoding the constant regions to lead to a preferentialpairing of the binding sites formed by the variable regions whenattached to the constant regions.

Antibodies are intended to refer to all variations of immunoglobulinsthat retain specific binding, such as Fabs, Fab′2, scFvs, but typicalfor antibodies described herein is the presence of a pair of amino acidsequences (at least two CDRs) that are paired to form a binding site.Thus, the also provided is a method wherein the variable regions arederived from heavy chains and/or light chains of immunoglobulins,engineered versions of variable regions with elements of heavy and/orlight chains of immunoglobulins and/or a method wherein theproteinaceous molecules are antibodies, fragments and/or derivatives ofantibodies.

The methods provided are typically for the production of multiple (i.e.,three or more) binding specificities in one system. Because of thespecific design of the contributing variable regions this has now becometechnically and commercially feasible.

Production may be controlled by placing the expression of differentvariable regions under control of different elements such as promoters,(trans) activators, enhancers, terminators, anti-repressors, repressors,and the like. These control elements may be inducible or repressible.Thus, the production of variable regions can be regulated, thusoptimizing pairing conditions as desired. Different combinations ofvariable regions can be made by separation in time of expression ofvarious variable regions and/or ratios between different paired variableregions may be manipulated by regulating expression levels. Variationsare described in the detailed description. Also provided is anexpression system comprising nucleic acids encoding variable regionstogether with all elements required for gene expression and pairing,such an expression system may comprises at least one recombinant cell,such as a bacterium, a yeast cell, a fungal cell, an insect cell, aplant cell or another eukaryotic cell, in particular, a mammalian cell,more in particular, a human cell or a mouse cell.

Such a system can be provided with all necessary and useful controlelements as disclosed herein before and as well known in the art.Selection elements and suicide elements may also be introduced into sucha system as desired.

A collection of expression systems comprising a variety of combinationsof different specificities is also provided, typically as a library foruse in selecting desired combinations of variable regions.

Also disclosed are methods and means for producing molecules comprisingvariable regions that are specific for their targets, but are lessimmunogenic. Described herein, the reduction of immunogenicity is atleast partially achieved by providing a nucleic acid molecule encodingat least an immunoglobulin light chain or heavy chain, wherein theheavy- or light chain encoding sequence is provided with a means thatrenders it resistant to DNA rearrangements and/or somatichypermutations. In certain embodiments, the nucleic acid molecule can beprovided cells that are used to generate a transgenic non-human mammal.In other embodiments, the nucleic acid molecule can be provided to othercells from such a transgenic non-human animal The non-human mammal maybe, e.g., a rodent, or, more specifically, may be a mouse. In certainembodiments, the nucleic acid encodes a human, human-like, or humanizedimmunoglobulin chain

In the remainder of this specification, mice are typically used asexamples of non-human mammals The transgenic, non-human, mammalian hostsare capable of mounting an immune response to an antigen, where theresponse produces antibodies having primate, particularly human,variable regions. Various transgenic hosts may be employed, particularlymurine, lagomorpha, ovine, avine, porcine, equine, canine, feline, orthe like. Mice have been used for the production of B-lymphocytes forimmortalization for the production of antibodies. Since mice are easy tohandle, can be bred in large numbers, and are known to have an extensiveimmune repertoire, mice will usually be the animal of choice. Therefore,in the following discussion, the discussion will refer to mice, but itshould be understood that other animals, particularly non-primatemammals, may be readily substituted for the mice, following the sameprocedures.

A reason for preventing rearrangements and hypermutation is that in thismanner, a non-immunogenic polypeptide can be chosen beforehand knowingthat this polypeptide chain will remain non-immunogenic. At least one ofthe chains of the resulting immunoglobulin is thus less immunogenic. Theresulting antibody needs to have (usually) both a light- and a heavychain The non-immunogenic chain must therefore be capable of pairingwith the other chain The other chain may be an endogenous chain, anexogenous chain or a hybrid of both. For human therapy, thenon-immunogenic chain should be as close to human as possible.

A means for rendering a gene encoding an immunoglobulin chain (orchains) resistant to DNA rearrangement and/or mutation is removal of allgenetic elements responsible for the rearrangement and/or mutation. Thedrawback thereof is that the variability of the two chains iseliminated, whereas the transgenic non-human mammal may retain thevariability in one chain (for example the heavy chain) and inhibitsand/or prevents the rearrangement-mutation of the other chain (forexample the light chain).

The elements for rearrangement and/or hypermutation characterized so farare located within the loci for immunoglobulins. Therefore, the meansfor rendering the immunoglobulin encoding sequence resistant to DNArearrangement and/or mutation is inserting the gene in a locus outsidethe immunoglobulin loci.

Thus, as described herein, a transgenic non-human mammal is providedwherein the light/heavy chain encoding sequences are integrated in thegenome of the non-human mammal in a locus outside the immunoglobulinloci. In certain embodiments, the insertion is in a locus that isresistant to gene silencing. As described herein, the integration may bein the Rosa-locus or a comparable locus.

In certain embodiments, provided is an expression cassette that can beinserted into a Rosa locus of the non-human animal or comparable locuswith a means that allows expression of the immunoglobulin chain(s)essentially limited to cells of B cell lineage, for example with a meansthat allows expression of the light chain encoding nucleic acid during acertain stage of the development of B cells. The term “essentiallylimited expression” indicates that expression is predominantly in cellsof the B-cell lineage, but that lower levels of expression in othercells, as compared to the level of expression in B-cells, is possible.In certain embodiments, the term “essentially limited expression”indicates that the expression is exclusively present in cells of theB-cell lineage. Such means typically include B cell (developmentalstage) specific promoters such as CD19, CD20, μHC (all V-genes), VpreB1,VpreB2, VpreB3, λ5, Igα, Igβ, κLC (all genes), λLC (all genes), BSAP(Pax5). Although it is very well possible to direct the expression ofthe DNA rearrangement and/or mutation resistant chain by such promoters,they are relatively weak. A strong promoter will typically be requiredto ensure adequate surface expression of the B cell receptor (made up ofthe membrane attached Ig H and L chain) and to compete with theexpression and pairing of endogenous chains (if present) through allelicexclusion. Such a promoter, however is usually not tissue specific. Toconfer tissue specificity, an indirect system employing Cre/lox or thelike may be sued. The desired chain is put under control of a strongpromoter inhibited by an element that can be removed by the action of aCre-protein, leading to activation of the desired immunoglobulinencoding gene. This system is described in detail in F. T. Wunderlich(2004), “Generation of inducible Cre systems for conditional geneinactivation in mice,” Inaugural dissertation zur Erlangung desDoktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät derUniversität zu Köln; on the internet atdeposit.ddb.de/cgi-bin/dokserv?idn=97557230x&dok_var=dl&dok_ext=pdf&filename=97557230x.pdf.

In certain embodiments, the immunoglobulin chain produced in a mannerresistant to rearrangements and hypermutation is a light chain capableof pairing with different heavy chains encoded by the non-human mammalThe light chain will then be the same (and less immunogenic) in allantibodies, but variety in specificity is retained throughrearrangements and hypermutations in the heavy chains. It may in thatcase be desirable to silence at least one of the endogenous lociencoding a light chain, although allelic exclusion may render thisunnecessary.

According to this embodiment, the endogenous kappa (κ) light chain locusmay be functionally silenced.

If the endogenous κ light chain locus is silenced, but also for otherreasons, it is desireable that the resistant light chain is a κ lightchain, desireably a light chain that has a germline-like sequence. Asdescribed herein, such a light chain would lead to an antibody withreduced immunogenicity. One example of a germline sequence is based onthe human IGKV1-39 (O12) as this light chain is very frequently observedin the human repertoire (de Wildt et al. 1999, J. Mol. Biol. 285(3):895and has superior thermodynamic stability, yield and solubility (Ewert etal. 2003, J. Mol. Biol. 325(3):531).

The following description gives more specific embodiments of theexpression cassette with which the non-human animal can be provideddescribed herein. Although this is typically advantageous forimmunoglobulins, other genes of interest are also contemplated.

Thus, provided in a specific embodiment is a transgenic non-human mammalwherein the light chain encoding nucleic acid comprises in 5′-3′direction: a B cell specific promoter, a leader, a rearranged human Vgene, optionally a MoEκi enhancer, a constant region (κ) and optionallya (truncated) MoEκ3′ enhancer. Neuberger identified and examined a novelB-cell specific enhancer located downstream of the kappa constant region(EPO patent application EP004690251, the contents of which areincorporated herein by this reference). This enhancer has been shown toplay a crucial role in the expression of kappa genes as removal of the808 bp enhancer strongly reduced expression. Deletion of the 3′ kappaenhancer also strongly reduced the level of somatic hypermutations(SHM). In transgenic and cell expression studies it has been revealedthat reduced, mutated or deleted 3′ kappa enhancers not only loweredexpression levels but also decreased the level of somatichypermutations. Currently, it cannot be determined whether the 3′ kappaenhancer is involved in SHM processes, expression regulation or both(review V. H. Odegard et al. (2006), Nat. Rev. Immunol. 6:573; M. Inlayet al. (2002), Nat. Immunol. 3:463.).

Detailed expression studies using engineered variants of the 3′ kappaenhancer indicated that a 50 nucleotide region is sufficient to driveexpression. However for proper expression a reduced sequence of 145nucleotides may be used (EP04690251; K. B. Meyer et al. (1990), NucleicAcids Res. 18(19):5609-15).

Thus, in one aspect is provided a nucleic acid for insertion into thegenome of a non human animal that is an expression cassette for theexpression of a desired proteinaceous molecule in cells developing intomature B cells during a certain stage of development, the cassettecomprising means for preventing silencing of expression of the desiredproteinaceous molecule after introduction into a host cell, and meansfor timing expression of the desired proteinaceous molecule with thedesired developmental stage of the host cell.

An expression cassette is defined as a nucleic acid molecule that hasbeen provided with all elements necessary for expression of the gene ina host cell, although in certain embodiments some of such elements maybe present on a second nucleic acid to be introduced, whereby theseelements act in trans. Elements necessary for expression in a host cellinclude promoters, enhancers and other regulatory elements. Only thoseelements are necessary that are not provided by the host cell. Further,an expression cassette may comprise means for introduction into thegenome of a host cell, such as sequences that allow for homologousrecombination with a certain site in the genome. Usually, the nucleicacid moledule will be DNA, typically double stranded. Typically, theexpression cassette will be provided to the cell in a vector from whichit is transferred to the genome of the cell.

The expression of a gene of interest should not be silenced in thegenome of the host cell, especially not in the development stage whereexpression is required. This can be done by various means, such asinsertion into the endogenous locus or by providing the cassette withnucleic acid elements that prevent silencing (Kwaks et al. (2006),Trends Biotechnol. 24(3):137-142, which is incorporated herein byreference). In certain emobidments, the expression cassette is insertedin a locus that is not silenced in the host cells (EP 01439234; which isincorporated herein by reference).

Means for preventing silencing comprise Stabilizing Anti-Repression-sequences (STAR®-sequences) and Matrix Attachment Regions (MARs). A STARsequence is a nucleic acid molecular sequence that has a capacity toinfluence transcription of genes in cis. Typically, although notnecessarily, a STAR sequence does not code by itself for a functionalprotein element. In one embodiment, one STAR element is used. In certainembodiments, however, more than one STAR element is used. In aparticular embodiment, an expression cassette described herein isprovided with two STAR sequences; one STAR sequence at the 5′ side ofthe coding sequence of the immunoglobulin gene and one STAR sequence atthe 3′ side of the coding sequence of the immunoglobulin gene. MARs areDNA sequences that are involved in anchoring DNA/chromatin to thenuclear matrix and they have been described in both mammalian and plantspecies. MARs possess a number of features that facilitate the openingand maintenance of euchromatin. MARs can increase transgene expressionand limit position-effects.

In certain embodiments, expression from the cassette may only occurduring a certain period in the development of a cell, in particular adeveloping B cell, more in particular a B cell in a transgenic non-humananimal, in particular a mouse. The developmental period may be chosensuch that the expression of the gene from the cassette (typically alight- or heavy chain-like polypeptide) does not significantly interferewith the normal differentiation and/or maturation of the cell and whenapplicable, allows for pairing of the polypeptide chain produced withits counterpart.

This may, in one embodiment, be achieved by providing a nucleic acidmolecule described herein, wherein the means for timing expression is apromoter of which the activity is essentially limited to the certainstage of development. In a developing B cell, which, e.g., afterimmunization is maturing and/or differentiating, the expression of thegene of interest, when it is one of the polypeptide chains of animmunoglobulin, must not interfere (significantly) with the maturationand/or differentiation and it needs to be timed such that the resultingpolypeptide can pair with its counterparts. Therefore, provided is anucleic acid molecule as described herein wherein the certain stagestarts at a stage immediately preceding or coinciding with the onset ofthe expression of light chain molecules by the cells at a certain stageof development into a mature B cell. This may be achieved by selecting apromoter that is active only during the suitable period. Such a promotermay be a CD19 promoter, the Ig-a promoter, the Ig-β promoter, the μhc(all genes) promoter, the Vk promoter or analogues or homologues thereof

In a specific embodiment, the promoter as disclosed herein does notdrive the expression of the gene of interest directly. Instead, itdrives the expression of a gene of which the product activates in transthe expression of the gene of interest. Such an activating gene may be agene encoding a so-called Cre recombinase or Cre-like protein. Theexpression cassette for the gene of interest may, e.g., be provided witha sequence that inhibits expression of the gene of interest. Thesequence can be removed by the action of the Cre recombinase, which isunder control of the desired promoter (active during the proper stage ofdevelopment). In this embodiment a set of expression cassettes isrequired.

Therefore, provided is a set of nucleic acid molecules that areexpression cassettes, wherein one nucleic acid molecule comprises anexpression cassette encoding a Cre-like protein under control of apromoter active during the desired stage of development of the host celland the second nucleic acid comprises a sequence encoding a desiredproteinaceous molecule under control of a constitutive promoter whichcan be activated by the action of a Cre-like protein. The activation maybe achieved by removal of a stop sequence flanked by loxP sites. TheCre/lox system is described in detail in Rajewsky et al. (1996), J.Clin. Invest. 98:600-603, which is incorporated herein by reference.Such systems are reviewed in F. T. Wunderlich (2004), “Generation ofinducible Cre systems for conditional gene inactivation in mice,”Inaugural dissertation zur Erlangung des Doktorgrades derMathematisch-Naturwissenschaftlichen Fakultat der Universitat zu Koln;on the World Wide Web atdeposit.ddb.de/cgi-bin/dokserv?idn=97557230x&dok_var=dl&dok_ext=pdf&filename=97557230x.pd,which is incorporated herein by reference.

Further provided is a transgenic non-human animal that has been providedwith an expression cassette hereof, wherein the desired proteinaceousmolecule is a polypeptide chain of an immunoglobulin. One example of apolypeptide chain is a light chain. A more specific example of apolypeptide is a germline or germline-like light chain An even morespecific polypeptide is O12. In certain embodiments, the rearrangedgermline kappa light chain IGKV1-39*01/IGKJ1*01 (nomenclature accordingto the IMGT database, at [worldwideweb].imgt.org).

In certain embodiments, the polypeptide chain is rendered essentiallyincapable of rearrangement and/or of excluded of any sequencemodification such as normally operating on Ig during the process of Bcell affinity maturation. Therefore, provided is a transgenic non-humananimal that has been provided with an expression cassette describedherein, wherein the rearrangement and/or sequence modifications areprevented by the absence of elements at least partially responsible forsomatic hypermutation such as, for example, the MoEκi enhancer.

One example of an expression cassette described herein comprises meansfor prevention of silencing. In one embodiment, the means for preventionof silencing are means for insertion into a locus in the genome of thehost cell that is resistant to silencing. The means for insertion may bemeans for homologous recombination into the site resistant to silencing.An example locus when the non-human animal is a mouse is the rosa-locus.

A further example of an expression cassette described herein comprisesin 5′-3′ direction: a Vκ promoter, a mouse leader, a human V gene,optionally a MoEκi enhancer, a rat constant region (Cκ) and optionally a(truncated) MoEκ3′ enhancer.

Yet a further example of an expression cassette described hereincomprises in 5′-3′ direction: a Vκ promoter, a human leader, a human Vgene, optionally a MoEκi enhancer, a rat constant region (Cκ) andoptionally a (truncated) MoEκ3′ enhancer.

Certain antibodies produced as described herein may be be used in humantherapeutics and diagnostics. Thus, provided is a method for producing adesired antibody comprising exposing a non-human mammal described hereinto an antigen such that an antibody response is induced and isolatingthe antibodies specific for the antigen.

In certain embodiments, provided are methods for producing a desiredantibody, the method comprising exposing a non-human mammal describedherein to an antigen such that an antibody response is induced andisolating cells producing such antibodies, culturing and optionallyimmortalizing the cells and harvesting the antibodies.

In certain embodiments, provided is a method for producing a desiredantibody comprising exposing a non-human mammal described herein to anantigen such that an antibody response is induced and isolating anucleic acid encoding at least part of such an antibody, inserting thenucleic acid or a copy or a derivative thereof in an expression cassetteand expressing the antibody in a host cell.

The methods for producing antibodies from transgenic mice are known to aperson skilled in the art. Examples are methods for production ofmixtures of antibodies from one cell, whereby the nucleic acids encodingthese antibodies have been derived from mice described herein.

These so-called OLIGOCLONICS® are disclosed in WO 04106375 and WO05068622, which are incorporated herein by reference.

Described herein are transgenic non-human mammals, such as mice, capableof generating specific and high affinity hybrid mouse-human antibodieswith, for example, human immunoglobulin light chain variable (VL)regions in or near germline configuration and, for example, murineimmunoglobulin heavy chain variable (VH) regions that may haveaccumulated somatic mutations during the process of antigen-drivenaffinity maturation. It is envisaged that the murine VH regions of thehybrid antibodies may be subjected to humanization procedures to yieldmAbs that have reduced immunogenicity when applied in humans based ongermline or near-germline VL regions and murine VH regions that havebeen humanized

In particular, shown is that transgenic mice that harbor a DNAexpression construct that encodes a rearranged human VL region under thecontrol of cis-acting genetic elements that provide timely and regulatedexpression of the transgene on a significant proportion of B cellsduring B cell development, yet lack elements that direct the somatichypermutation machinery to the transgene, are capable of generatingspecific and high affinity mouse-human hybrid antibodies withessentially unmutated L chains It is shown that the rearranged humantransgene is capable of pairing with a diversity of endogenous murineimmunoglobulin H chains to form mouse-human hybrid immunoglobulinsexpressed on the surface of B cells and to sufficiently facilitatemurine B cell development to obtain a sizeable and diverse peripheral Bcell compartment.

In certain embodiments, the transgene expression construct harbors thecoding sequences of a human rearranged L chain V region under thecontrol of a human VL promoter to direct B-cell specific expression. Inaddition, the construct harbors the murine 3′ Ck enhancer sequence for Bcell specific and inducible and high level expression of the transgene.Furthermore, the construct is designed to lack regulatory elements thatfacilitate the recruitment of the somatic hypermutation machinery to thetransgene, such as the intron enhancer and the 3′ C-kappa enhancer.

In a related embodiment, the rearranged human VL gene is inserted in themurine Rosa26 locus by site-specific integration. The Rosa26 locus isuseful in the context of the “targeted transgenesis” approach forefficient generation of transgenic organisms (such as mice) with apredictable transgene expression pattern.

In certain embodiments, the rearranged human VL region is selected forits capacity to pair with many different murine VH genes so as to ensurethe generation of a population of B cells with a diverse VH generepertoire. A method of obtaining such VL regions comprises amplifying arepertoire of rearranged VH genes from the B cells of mice and arepertoire of human rearranged germline VL regions from the B cells ofhumans and cloning them into phagemid display vectors to prepare diverselibraries of hybrid immunoglobulins in bacteria. By nucleotide sequenceanalysis of collections of unselected and antigen-selected VH/VL pairs,human germline VL genes that pair with many different murine VH genesare identified. A collection of human germline VL genes with thiscapacity is described.

In one embodiment, it is shown that upon immunization with antigen, theB cells are capable of mounting an immune response, leading to thegeneration of B cells that secrete hybrid antibodies with highspecificity and affinity. The V regions encoding these antibodies arecharacterized by the human transgenic light chain that harbors no orvery few mutations and a murine heavy chain that harbors a variablenumber of mutations introduced by the somatic hypermutation machinery.

In a related embodiment, strategies to obtain high affinity hybridmonoclonal antibodies from the transgenic mice by hybridoma and displaytechnologies are contemplated as well as procedures to humanize themurine VH regions to obtain less immunogenic antibodies for applicationin humans.

In one embodiment, provided is an immunoglobulin L chain transgeneconstruct comprising DNA sequences that encode a human immunoglobulin VLregion in combination with a light chain constant region (CL) of ananimal immunoglobulin protein, which sequences are operably linked totranscription regulatory sequences that, when integrated in a non-humantransgenic animal, produce an Ig VL-CL polypeptide with a human VLregion that is not or marginally subject to somatic hypermutation. TheIg VL is capable of pairing with rearranged VH-CH polypeptides that aregenerated during B cell development in the non-human transgenic animal,with the VH-CH polypeptides retaining the capacity to undergo somatichypermutation upon stimulation. The CL region may be of any animalspecies and is generally capable of pairing with the CH regions of thenon-human transgenic animal

Also included is the use of a transgene construct as above in producinga transgenic non-human animal capable of the production of hybridantibodies consisting of VL-CL polypeptides and VH-CH polypeptides inwhich the VL region is of human origin and the CL, VH and CH may be ofany animal species, including human. Upon immunization, these transgenicanimals are capable of generating high affinity antibodies encoded bysomatically hypermutated VH genes and essentially non-mutated VL genesencoded by the transgene.

In another aspect, provided is a process for the production of atransgenic non-human animal capable of the production of hybridantibodies in response to antigenic challenge, comprising functionallydisrupting the endogenous immunoglobulin light chain locus and insertinginto the animal genome a transgene construct described herein.

Included is the use of animals obtainable by this process in theproduction of B cells that produce immunoglobulin having human VL lightchain. In another aspect there is provided a process for the productionof B cells that produce immunoglobulin having human VL and binding to aselected antigen, comprising challenging an animal obtainable by aprocess as above with the antigen and screening for B cells from theanimal that bind the antigen. Further included are B cells obtainable bythis process and hybridomas obtainable by immortalizing such B cells,e.g., hybridomas obtained by fusing B cells as above with myeloma cells.Also included is a process for producing monoclonal antibody comprisingcultivating such a hybridoma. In yet a further aspect, provided is theuse of the above B cells in producing a hybridoma or correspondingmonoclonal antibody.

Described herein is a process for the producing immunoglobulin(s) havinghuman VL chain and binding to a selected antigen, the process comprisingchallenging an animal obtainable as aforementioned with the antigen andobtaining immunoglobulin therefrom.

In one strategy, as an individual step, a rearranged VL region encodedby human germline V and J gene segments and a light chain constantregion of any animal species. For example, a murine constant region isintroduced into the mouse germ line. The transgene DNA may be introducedinto the pronuclei of fertilized oocytes or embryonic stem cells. Theintegration may be random or homologous depending on the particularstrategy to be employed. For example, the VL transgene may be introducedby random insertion, resulting in mice that bear one or multiple copiesof the transgene in the genome. Alternatively, the human VL transgenemay be targeted to a specific genomic locus using site-specificrecombination as described in the art.

In certain embodiments, the VL transgene is targeted to the murineROSA26 locus which is a suitable integration site allowing strong andpredictable expression of inserted transgenes (European Patent Officedocument EP 1,439,234 A1, the contents of which are incorporated hereinby this reference). The targeting vector allows insertion of a singlecopy of a gene expression cassette, thus avoiding modulation oftransgene expression by the arrangement of multiple copies. By choosingthe autosomal Rosa26 locus as insertion site, the expression pattern ofthe inserted transgene in the non-human animal is predictable.Furthermore, random X inactivation and/or modulation by chromosomalposition effects are avoided. This also eliminates the need to generateand analyze multiple transgenic strains for any given transgene.Finally, the Rosa26 targeting vector for the site-specific integrationcan be used for multiple gene expression cassettes. Thus, it may beenvisaged that two or more different rearranged germline human VLregions are inserted into the Rosa26 locus to further increase thediversity of the repertoire of hybrid or human antibodies.

In another embodiment, a rearranged human VL region may be targeted tothe murine Ig kappa or lambda light chain locus so as to functionallyinactivate the endogenous locus or mice containing the rearranged humanVL region may be bred with mice that lack functional kappa or lambda Igloci or both. Thus, by using transformation, using repetitive steps orin combination with breeding, transgenic animals may be obtained whichare able to produce antibodies harboring the human VL transgene in thesubstantial absence of endogenous host immunoglobulin light chains

In one embodiment, a human VL transgene is selected for its capacity topair with a substantial portion of murine VH regions to form a diverserepertoire of functional mouse-human hybrid antibodies expressed on thesurface of B cells. By a substantial portion of murine VH regions ismeant that the human VL pairs with at least with 0.1%, 1%, or 10% of themurine VH regions generated during B cell development Methods toidentify human VL genes with this characteristic include randomlypairing a repertoire of human VL regions with a repertoire of murine VHregions, co-expression of VH and VL regions in appropriate eukaryotic orprokaryotic expression vectors and screening for human VL regions thatpair with a substantial portion of murine VH regions. In one embodiment,phagemid vectors may be used to direct expression of mouse-humanantibody fragments in bacterial cells or to the surface of filamentousphage and analysis of binding capacity of antibody fragments by methodsknown in the art.

In another embodiment, a human VL transgene is selected for its capacityto pair with a substantial portion of human VH regions to form a diverserepertoire of human antibodies expressed on the surface of B cells. By asubstantial portion of human VH regions is meant that the human VL pairswith at least with 0.1%, 1%, or 10% of the human VH regions generatedduring B cell development

In the latter embodiment, the human VL transgenic mice are crossed withmice that harbor functional rearranged or non-rearranged human H chainimmunoglobulin loci and functionally inactivated endogenous H chain Igloci as described in the art. The functional inactivation of the twocopies of each of the three host Ig loci (heavy chain, kappa and lambdalight chain), where the host contains the human IgH and the rearrangedhuman VL transgene would allow for the production of purely humanantibody molecules without the production of host or host human chimericantibodies. Such a host strain, by immunization with specific antigens,would respond by the production of mouse B-cells producing specifichuman antibodies, which B-cells are subsequently fused with mousemyeloma cells or are immortalized in any other manner for the continuousstable production of human monoclonal antibodies. Alternatively, thepopulation of B cells is used as a source of VH regions that can beobtained by constructing cDNA libraries or by PCR amplification usingprimers for human VH regions as is known in the art.

A human rearranged VL gene is reconstructed in an appropriate eukaryoticor prokaryotic microorganism and the resulting DNA fragments can beintroduced into pronuclei of fertilized mouse oocytes or embryonic stemcells. Various constructs that direct B cell specific expression of VLtransgenes have been described in the art and have the following generalformat: a leader sequence and relevant upstream sequences to direct Bcell specific expression of the transgene, a coding sequence of a humanVL transgene, an enhancer sequence that directs B cell specific and highlevel expression of the transgene and a murine constant region gene. Inone format, the enhancer is the C-kappa 3′ enhancer because it directshigh level expression in B-lineage cells, but does not recruit somatichypermutation when used in transgene constructs.

In one embodiment, animals, for example, mice, comprising one ormultiple copies of the transgene in the genome are isolated and analyzedfor stable expression Animals are selected that show stable expressionof the transgene over longer periods of time, for example in B-cells. Ifrequired, different animal lines comprising independent insertions ofone or multiple copies of the transgene, for example on differentchromosomes, are crossed to obtain animals with different insertions ofone or multiple copies of the transgene to increase expression of thetransgene in animals, for example in B-cells.

Further provided is progeny of a transgenic non-human animal describedherein, the progeny comprising, at least in its B-cell lineage, a heavy-or light chain encoding sequence together with a means that renders thesequence resistant to DNA rearrangements and/or somatic hypermutations.

Further provided is progeny of a transgenic non-human animal describedherein, the progeny comprising an expression cassette for the expressionof a desired proteinaceous molecule in cells during a certain stage ofdevelopment in cells developing into mature B cells.

In addition, provided is a cell that is isolated from a transgenicnon-human animal described herein, the cell comprising a heavy- or lightchain encoding sequence together with a means that renders the sequenceresistant to DNA rearrangements and/or somatic hypermutations. Inaddition, provided is a cell that is isolated from a transgenicnon-human animal described herein, the cell comprising an expressioncassette for the expression of a desired proteinaceous molecule in cellsduring a certain stage of development in cells developing into mature Bcells. A cell described herein, for example an antibody-producing B-cellor a cell that is capable of differentiating or maturating into anantibody-producing B-cell, can be used for in vitro production ofantibodies, as is known to the skilled person, for example, from Gascanet al. 1991, J. Exp. Med. 173:747-750. Methods for immortalization of acell described herein are known in the art and include the generation ofhybridomas, for example, by fusion with a myeloma cell, transformationwith Epstein Barr Virus; expression of the signal transducer ofactivation and transcription (STAT), activation via CD40 and IL4receptor signaling, and/or expression of Bcl6 (Shvarts et al. 2002,Genes Dev. 16:681-686).

In a separate step, the endogenous Kappa and Lambda light chain loci arerendered essentially non-functional such that at least the majority of Bcells in the transgenic mice bear Ig receptors that contain thetransgenic human VL region. Inactivation of the endogenous mouseimmunoglobulin loci is achieved by targeted disruption of theappropriate loci by homologous recombination in mouse embryonic stemcells. The targeted disruption comprises alteration of the genomicsequence such that substantially no functional endogenous mouseimmunoglobulin Kappa and/or Lambda light chain is produced. The term“substantially no functional endogenous immunoglobulin” indicates thatthe endogenous Kappa and/or Lambda light chain loci are functionallysilenced such that the level of functional protein expression of theendogenous Kappa and/or Lambda light chain loci is reduced to about 20%,about 10%, about 5%, about 2% or about 1% of the level of expression ina reference mouse. In one embodiment, the level of functional proteinexpression of the endogenous Kappa and/or Lambda light chain loci isreduced to 0%. The level of functional protein expression can bedetermined by means known to the skilled person, including westernblotting and pairing with a mouse heavy chain The reference mouse is amouse in which the endogenous Kappa and/or Lambda light chain loci isnot disrupted. The alteration comprises mutation and/or deletion of genesequences that are required for functional expression of the endogenousimmunoglobulin genes. Alternatively, the alteration comprises insertionof a nucleic acid into the endogenous mouse immunoglobulin Kappa and/orLambda light chain loci such that the functional expression of theendogenous immunoglobulin genes is reduced. In one embodiment, thenucleic acid comprises a silencing element resulting in transcriptionalsilencing of the endogenous immunoglobulin gene. In a furtherembodiment, or in addition, the nucleic acid comprises a sequence thatdisrupts splicing and/or translation of the endogenous immunoglobulingene, for example, by introducing an exon that renders a frame shift inthe coding sequence, or that comprises a premature stop codon. In eachcase chimeric animals are generated which are derived in part from themodified embryonic stem cells and are capable of transmitting thegenetic modifications through the germ line. The mating of mouse strainswith human immunoglobulin loci to strains with inactivated mouse lociyields animals which produce antibodies comprising essentially onlyhuman light chains.

A construct for homologous recombination is prepared by means known inthe art and any undesirable sequences are removed, e.g., procaryoticsequences. Any convenient technique for introducing a construct forhomologous recombination into a target cell may be employed. Thesetechniques include spheroplast fusion, lipofection, electroporation,calcium phosphate-mediated DNA transfer or direct microinjection. Aftertransformation or transfection of the target cells, target cells areselected by means of positive and/or negative markers, for example, byneomycin resistance and/or acyclovir and/or gancyclovir resistance.Those cells which show the desired phenotype may then be furtheranalyzed by restriction analysis, electrophoresis, Southern analysis,PCR, or the like. By identifying fragments which show the presence ofthe lesion(s) at the target locus, cells in which homologousrecombination has occurred to inactivate a copy of the target locus areidentified.

Furthermore, it is shown that upon immunization, the murine and human VHregions in the afore-mentioned transgenic mice but not the VL regionsare capable of undergoing somatic hypermutations to generate highaffinity antibodies. Advantageously, these antibodies encoded bygermline VL regions are predicted to contribute to lower immunogenicitywhen applied in humans and result in more stable antibodies that areless prone to aggregation and thus safer for therapeutic use in humans.

MAbs derived from the afore-mentioned non-human transgenic animals orcells all share the same identical human VL regions. It has beendescribed that mAbs that share the same identical VL region may beco-expressed in a single clonal cell for the production of mixtures ofrecombinant antibodies with functional binding sites (see, theincorpoarated WO 04106375 and WO 05068622). Thus, provided is a platformfor the generation of specific and high affinity mAbs that constitutethe basis for mixtures of mAbs produced by clonal cells.

In certain embodiments mAbs derived from the afore-mentioned non-humantransgenic animals or cells are directed against cellular targets.Examples of targets are human surface-expressed or soluble proteins orcarbohydrate molecules. Further, examples of targets aresurface-expressed proteins or carbohydrate molecules that are expressedon the surface of bacteria, viruses, and other pathogens, especially ofhumans.

More specifically, example targets include cytokines and chemokines,including but not limited to InterLeukin 1beta (IL1beta), IL2, IL4, IL5,IL7, IL8, IL12, IL13, IL15, IL18, IL21, IL23 and chemokines such as, forexample, CXC chemokines, CC chemokines, C chemokines (or y chemokines)such as XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β), and CX3Cchemokines. Further examples of targets are receptor molecules of thecytokines and chemokines, including type I cytokine receptors such as,for example, the IL-2 receptor, type II cytokine receptors such as, forexample, interferon receptors, immunoglobulin (Ig) superfamilyreceptors, tumor necrosis factor receptor family including receptors forCD40, CD27 and CD30, serine/threonine-protein kinase receptors such asTGF beta receptors, G-protein coupled receptors such as CXCR1-CXCR7, andtyrosine kinase receptors such as fibroblast growth factor receptor(FGFR) family members, EGF receptor family members including erbB1(EGF-R; HER1), erbB2, (HER2), erbB3 (HER3), and erbB4 (HER4), insulinreceptor family members including IGF-R1and IGF-RII, PDGF receptorfamily members, Hepatocyte growth factor receptor family membersincluding c-Met (HGF-R), Trk receptor family members, AXL receptorfamily members, LTK receptor family members, TIE receptor familymembers, ROR receptor family members, DDR receptor family members, KLGreceptor family members, RYK receptor family members, MuSK receptorfamily members, and vascular endothelial growth factor receptor (VEGFR)family members.

Further example targets are targets that are over-expressed orselectively expressed in tumors such as, for example, VEGF, CD20, CD38,CD33, CEA, EpCAM, PSMA, CD54, Lewis Y, CD52, CD40, CD22, CD51/CD61,CD74, MUC-1, CD38, CD19, CD262 (TRAIL-R2), RANKL, CTLA4, and CD30;targets that are involved in chronic inflammation such as, for example,CD25, CD11a, TNF, CD4, CD80, CD23, CD3, CD14, IFNgamma, CD40L, CD50,CD122, TGFbeta and TGFalpha.

Surface-expressed proteins or carbohydrate molecules that are expressedon the surface of bacteria, viruses, and other parasitic pathogens,especially of humans, include surface markers of influenza A and Bviruses such as hemagglutinin (HA) and neuraminidase (NA), filovirusessuch as Ebola virus, rabies, measles, rubella, mumps, flaviviruses suchas Dengue virus types 1-4, tick-borne encephalitis virus, West Nilevirus, Japanese encephalitis virus, and Yellow fever virus,Paramyxoviruses including Paramyxovirus such as Parainfluenza 1, 3,Rubulavirus such as Mumpsvirus and Parainfluenza 2, 4, Morbillivirus,and Pneumovirus such as Respiratory syncytial virus, Vaccinia, smallpox, coronaviruses, including Severe Acute Respiratory Syndrome (SARS)virus, hepatitis virus A, B and C, Human Immunodeficiency Virus, herpesviruses, including cytomegalovirus, Epstein Barr virus, herpes simplexvirus, and Varicella zoster virus, parvoviruses such as, for example,B19; Legionella pneumophila; Listeria monocytogenes; Campylobacterjejuni; Staphylococcus aureus; E. coli O157:H7; Borrelia burgdorferi;Helicobacter pylori; Ehrlichia chaffeensis; Clostridium difficile;Vibrio cholera; Salmonella enterica Serotype Typhimurium; Bartonellahenselae; Streptococcus pyogenes (Group A Strep); Streptococcusagalactiae (Group B Strep); Multiple drug resistant S. aureus (e.g.,MRSA); Chlamydia pneumoniae; Clostridium botulinum; Vibrio vulnificus;Parachlamydia pneumonia; Corynebacterium amycolatum; Klebsiellapneumonia; Linezolid-resistant enterococci (E. faecalis and E. faecium);and Multiple drug resistant Acinetobacter baumannii.

Additional examples of targets are IL-6 and its receptor, IL-6Ralpha,glycoprotein-denominated gp130, RSV, especially the surface proteins F,G and SH and non-structural proteins such as N and M, and receptortyrosine kinases, in particular erbBl (EGF-R; HER1), erbB2, (HER2),erbB3 (HER3), erbB4 (HER4), IGF-R1 and IGF-RII, c-Met (HGF-R).

Therefore, provided is a platform for the generation of specific andhigh affinity mAbs against the above mentioned targets that constitutethe basis for mixtures of mAbs produced by clonal cells. In certainembodiments, the specific and high affinity mAbs comprise mAbs that aredirected against different epitopes on at least one of the targets. In afurther embodiment, the specific and high affinity mAbs comprise mAbsthat are directed against different targets, such as, for example, oneor more members of the EGF-receptor family, including erbBl (EGF-R;HER1), erbB2, (HER2), erbB3 (HER3) and erbB4 (HER4).

Unless otherwise defined, scientific and technical terms used inconnection with this disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See, e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (3rd edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)), which isincorporated herein by reference. The nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

Selection methods are also described herein. In one embodiment, providedis a method for selecting combinations of proteinaceous molecules havingspecific affinity for at least two target epitopes, the methodcomprising contacting a collection with the two target epitopes andselecting combinations showing the specific affinity.

Such methods are particularly useful when the two target epitopes areassociated with one disease or disorder. In certain embodiments, it ispossible to combine such a method with subjecting a selected combinationof proteinaceous molecules to a biological assay indicative of an effectof the combination on the disease and/or disorder.

Compositions obtainable by the methods described herein are alsoincluded. Examples are compositions comprising at least three differentpaired variable regions, having different binding specificities, inparticular, those wherein the variable regions are derived fromimmunoglobulin light chains and/or immunoglobulin heavy chains. Acombination composition that targets both TNF-α as well as IL-1β is anexemplary combination. In such typical therapeutic uses, it is importantthat the combination preparations do not lead to severe immune responsesin the subject to be treated. At least some of the antigenic parts ofthe binding molecules, such as the constant regions in antibodies shouldbe of human origin. In the alternative, antigenic parts may be omittedor masked by molecules such as PEG. Thus, also provided in oneembodiment is a composition, which is a pharmaceutical composition.Although antibodies have found use in other areas, and antibodycombinations as described herein can be used in other areas, such as thepharmaceutical use of the combinations, both diagnostic and therapeutic.However, in industrial applications the combinations may also besuperior to existing separation techniques, because of ease ofproduction, consistency of production and the availability of manycombinations of specificities, capable of separating almost anythingfrom any mixture. In testing, be it in pharmaceutical diagnostics or inany other field (environmental, agricultural, to name a few) thecombinations can be used advantageously as well. Both partners of asandwich assay can be made in one cell. Agglutination mixtures can bemade in one cell. When using the IgG format, the expression in the samecell will lead to a substantial fraction of bispecific compounds, whichoffer unique applications in combination with the monoclonals present inthe same mix. For example, when a monoclonal antibody can only bind withone arm to an antigen, a bispecific molecule with binding sites capableof binding to two different epitopes on the same antigen, may moreconsistently than the monoclonal antibody mixture immobilize or trapantigen. Again, ease and consistency of production, as well as thediversity of specificities is an asset of the combinations. Theseadvantages of course also apply in selecting and producing combinationsof specificities for therapeutic and/or prophylactic use, withadditional advantages in ease of selection, efficacy of selectedcombinations and the mentioned safety aspects.

A simple combination starts with two specificities present in thecombination. When a promiscuous variable region is present, such acombination requires only three different variable regions. Thecombination can be made such that all resulting paired variable regionsin one proteinaceous molecule have the same specificity, givingmonospecific molecules, or the variable or, if appropriate, the constantregions can be designed such that bispecific molecules are also present.It can also be designed such that one monospecific and one bispecificmolecule are present, but that the other possible monospecific moleculedoes not arise, because the variable regions cannot assemble in thatmanner Thus, one embodiment comprises a composition comprising at leastone monospecific antibody and at least one bispecific antibody producedin one cell for use as a pharmaceutical. In some applications,bispecific molecules, especially antibodies, may be advantageous forbringing two antigens together on a cell surface. Such aggregationevents are often required in biology for transduction of a signal to theinside of a cell. Bispecific antibodies in the mixture may also be usedto connect effector molecules with target cells. The uses envisaged forbispecific antibodies in the prior art are also envisaged for bispecificmolecules. The most advantageous compositions comprise more than twodifferent monospecific binding molecules, optionally together with thedifferent possible combinations of bispecific or multispecific moleculesthat may result from the different possible pairing events. Thesemultispecific mixtures resemble polyclonal mixtures in their efficacyfor recognizing antigens, but without the drawbacks of many irrelevantspecificities in the mixture. The mixtures resemble monoclonalantibodies in their defined constitution, ease of production and highspecificities, but without the concomitant loss of efficacy. Themixtures are referred to as OLIGOCLONICS®. OLIGOCLONICS® can thuscontain two, three, or more different binding specificities, and canexist in various formats. In the simplest form, OLIGOCLONICS® in the IgGformat contain a mixture of different monospecific antibodies andbispecific antibodies in a particular given ratio. In the Fab format,OLIGOCLONICS® contain a mixture of different Fab molecules which are theproduct of correctly paired variable regions. In the mixed format,OLIGOCLONICS® contain a mixture of antibodies and antibody fragments.

As disclosed herein, the methods and means in one embodiment are theproduction of combinations of specificities. Before production ofcombinations, suitable combinations must be designed and/or selected.These methods for designing and selection are also part of the presentdisclosure. Thus, in a further embodiment, provided is a method forproducing nucleic acids encoding variable regions for use in a methodfor production of combinations of specificities comprising synthesizingnucleic acids encoding variable regions, expressing the nucleic acidsand allowing the expression products to pair and selecting nucleic acidsencoding variable regions having desired pairing behavior.

In an alternative embodiment, provided is a method for producing nucleicacids encoding variable regions for use in a method for production ofcombinations of specificities comprising altering existing nucleic acidsencoding variable regions, expressing the nucleic acids and allowing theexpression products to pair and selecting nucleic acids encodingvariable regions having desired pairing behavior. Of course, bothmethods may be combined and/or repeated in any order. Synthesis,alteration and selection methods are disclosed in more detail in thedetailed description.

Nucleic acid molecules for use in producing combinations ofspecificities may be those encoding immunoglobulin polypeptides. Ofcourse, all types of immunoglobulins, especially antibodies (IgM, IgE,IgGs, etc.), but also fragments (scFv, Fab, single-domain, engineeredvariants) can be used. Variable regions can, for example, be derivedfrom either immunoglobulin heavy chain variable regions, orimmunoglobulin light chain variable regions, but can also be engineeredhybrids of heavy and light chain variable regions (with, for example,swapped CDR regions or FR regions). Variable regions can, for example,be obtained from hybridomas, by cloning from immune or non-immune donorsor can be synthetically constructed variable regions. Even hybrids canbe produced using nucleic acids and methods described herein. Forexample, hybrids with different yet functional binding sites can be madeby providing elements from different isotypes, for example, IgM and IgG,or IgM and IgA. It should be born in mind that T cell receptors resembleantibodies in many respects. Thus, the methods can also be appliedadvantageously with T cell receptors, their variable regions and theirencoding nucleic acids. The methods may be carried out usingimmunoglobulins having different chains (T cell receptors), especiallyantibodies having light chains and/or heavy chains or parts/derivativesthereof. Of course part and/or derivatives are such parts and/orderivatives that do have specific binding properties comparable toimmunoglobulins.

This means that variable regions should at least comprise an elementwhich resembles a complementarity-determining region of an antibody(CDR). In certain embodiments, the variable regions have more than aCDR, and/or a variable region resembles in size and physicochemicalproperties a VH or VL of an antibody. The detailed description describesusing antibodies as an exemplary embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Examples of composition of three or six proteinaceous moleculeswith three different binding specificities. The use of antibodies withappropriate pairing between the variable regions yields mixtures ofantibodies that are bispecific or monospecific and bivalent (top panel).By appropriate engineering to manipulate the pairing between thevariable regions, mixtures of only bispecifics or bivalent moleculesarise (left hand side panels). In the legend on the right panel (greybox) it is indicated that the three symbols, the circle, triangle andsquare, represent binding sites consisting each of variable regions.

FIG. 2: Method to identify antibodies with pairing-compatible elementsby empirical analysis of antibody variable region combinations.

FIG. 3: Antibodies with similar light or heavy chain by selection fromlibraries with restricted diversity. In this example of a Fab library,one of the antibody chains is identical in all library members (thewhite chain), while the others contain amino acid diversity.

FIG. 4: Different approaches to select antibodies with appropriatepairing behavior. (a) selection of Fab library with constant lightchain, and equivalent for Fab library with diversity in light chain onlyin (d); (b) selection of antigen-binding single-domain antibody fromheavy chain only library, and equivalent for VL in (e); (c) selection oflibrary of chimeric chains of VH and VL (in which, for example, some CDRelements are swapped).

FIG. 5: Selecting antibodies with pairing-compatible variable regions byre-shuffling one chain. Starting point of the method is a repertoire ofantibody binding sites, with paired variable regions, such as in thisexample, an Fab repertoire. Similarly single chain Fv libraries can beused. In a typical selection (top) the initially present pairing ofvariable regions is maintained throughout the iterative selectionprocess; in the selection followed by reshuffling (steps 1-3), one ofthe two variable regions (for example the heavy chain) of the pairs thathave been selected on antigen, is combined with partner domains (forexample light chains) derived from either the selected population orfrom the original population). After this the selection (step 4), andthe subsequent procedure are repeated. Eventually, individualantigen-reactive antibodies are identified by screening methods.

FIG. 6: Example of a competitive selection of antibodies with adesirable pairing behavior. The method involves the co-expression of oneor more competing antibodies (top, left) in the same host cell as amember of an antibody library (bottom, left). Depicted is the method forFab fragments, as described in the text. The result of the pairingopportunities of VHCH1 (white boxes) chain when co-expressed with twoother Fab fragments is depicted. The original combination of the VH withits cognate light chain (hatched box), will retain its original bindingaffinity for antigen and can thus be selected.

FIG. 7: Identifying antigen-specific antibodies by co-transfecting heavychain gene libraries with an invariant light chain gene and screeningthe resulting antibody mixtures for antigen reactive antibodies. Withevery cycle of transfection and screening, the diversity of the VHlibrary is reduced (at position *), to eventually yield a population ofantigen-reactive heavy chain variable genes. The numbers indicate thatsampling of a library of 10⁸ different heavy chains can be carried outby screening the wells of ten 96-well tissue culture with each 100clones per well.

FIG. 8: Identifying antigen-specific antibodies by transfectingsecretable heavy chain gene libraries, assembly with an invariant lightchain and screening the resulting antibody mixtures for antigen reactiveantibodies. With every cycle of transfection and screening, thediversity of the VH library is reduced (at position *), to eventuallyyield a population of antigen-reactive heavy chain variable genes.

FIG. 9: Screening antibody mixtures produced by the same host cell foroptimal bio-activity. Mixtures are made by transfecting heavy chaingenes encoding the antibodies of interest (here number is 10) togetherwith optimally paired light chain, followed by cloning of cell lines,selecting stably producing cell lines, and eventually screening theresulting antibody mixtures for optimal bio-activity.

FIG. 10: Examples of antibodies with cross-over domains. Heavy chaindomains (grey striped boxes) and light chain domains (white boxes).

FIG. 11: Ex vivo assembly of antibodies (A) and the universal antibodyconcept (B). Antibodies are produced as separate chains and thencombined to form a functional antibody. This is in particularlyinteresting when making mixtures of antibodies, as indicated in (B),where depending on the input of the chains and the separation of themixing reactions.

FIG. 12: Dependent expression of Ig chains. Chain-1 is typically theheavy chain, which is under control of a promoter (P). The IRES sequencelinks the expression of the heavy chain with that of a transactivator;this activates a responsive promoter to induce expression of Chain-2,typically the light chain (see, text for details).

FIG. 13: The sequence of pSCFV (SEQ ID NO:148 of the SEQUENCE LISTING,incorporated herein by this reference), a pUC119-based plasmid suitablefor stepwise cloning of antibody variable regions and expression of scFvfragments.

FIGS. 14A and 14B: Schematic depiction of plasmid pSCFV-3 (A) andpSCFV-3 with three cloned scFv fragments, in this case derived from theantibodies JA, JB and M57. The black box is a schematic depiction of thehistidine stretch; other C-terminal-based tags are also indicated. S,signal sequence; rbs, ribosome binding site; AMPr, ampicillin resistancegene (beta-lactamase).

FIG. 15: Schematic depiction of the eukaryotic expression vectorVHExpress as also described in (Persic et al. (1997) 187:9-18) exceptthat this variant has a CMV promoter; its use for cloning scFv fragments(top, indicated for antibody JA) such that the expression of scFv-Fcfusions is achieved.

FIG. 16: Sequence alignment of the three light chains amino acidsequences of antibodies JA (Kappa) (SEQ ID NO:110), and JB (SEQ IDNO:112) and M57 (SEQ ID NO:114) (both lambdas). The position of the CDRsis indicated.

FIGS. 17A and 17B: pFAB-display: Schematic depiction of pFAb-display(top), and indication of cloning of VLCL and VH regions; the polylinkerregion (below). Legend as in FIG. 14.

FIGS. 18A-18F: Mutagenesis of heavy chain variable region of the JAantibody (SEQ ID NO:109); underlined region was mutagenized. Otherregions known to be important for the interaction with the VL: theresidues at the positions marked in color (bottom) or with the boxesaround the JA-VH sequence are, alternatively, suitable for mutagenesis(based on data fromworldwideweb.biochem.unizh.ch/antibody/Structures/DimContacts/VHDimHistFrame.html).

FIG. 19: Outline of an expression vector for human monoclonal antibodiesin eukaryotic cells. CMV: CMV promoter; p(A): polyadenylation signal;Neo: neomycin resistance gene; Amp: ampicillin resistance gene.

FIG. 20: Outline of the expression cassette and expression vectors foruse with eukaryotic cells. The legend of the vector elements is depictedon the right. On the left hand side top panel are depicted, as examples,four eukaryotic expression cassettes for three antibody heavy chains andone light chain The elements found in an expression cassette for asingle antibody chain encoding gene or nucleic acid typically comprisesa promoter, a Leader sequence, an open reading frame encoding theantibody chain of interest, a polyadenylation region and terminator, allin operable configuration. Further sites/regions used for site-directedand in some cases homologous, recombination, are shown (are alsooptional; indicated on top of the first expression cassette). On thebottom panel is depicted an exemplary vector backbone used for insertionof the top panel cassette(s). This scheme displays the typical elementsof a eukaryotic expression vector, comprising a bacterial origin ofreplication (such as Col E1), a bacterial selection marker (B-Select,such as the ampicillin resistance gene), a eukaryotic selection marker(Select, such as gpt, neo, zeo, etc., see text; useful when stableintegration into the host cell's genome is envisaged), and additionaloptional elements such as a bacteriophage packaging region (for ss-DNAproduction, such as f1), and an optional amplification marker (such asDHFR). Optional are other expression controlling elements (such as BEs,STAR, LCRs, MARs and the like, see below) and IRES; these are includedin later figures.

FIG. 21: Schemes depicting different formats for the co-expression ofantibody chain encoding genes, exemplified here for the case in whichtwo antibodies that share a common light chain (not shown) have to beco-expressed. (A) The basic individual cassettes, as separate cassettesand cloned into separate expression vectors. (B) This cassette containsthe two Heavy chain (H) genes cloned in tandem, but their expression isindividually regulated, via two different promoters, P1 and P2. (C) Thetwo H genes are cloned into transcriptionally opposite directions and inthis example separated by an element that influences theexpression/stability/integration frequency (further examples are givenin the text). (D) Same as B, but now additional E-elements are includedat the 3′ end of each of the two transcriptional units. (E) For cases inwhich two binding proteins should be present in the mixture at roughlysimilar quantities, an IRES is inserted between two H genes. (F and G)Expression cassettes for mediating the expression of two H chains, inwhich each of the H genes are linked via an IRES element to a selectionmarker (which is then selected for instead of using thevector-backbone-based marker), without (G) or with (H) additionalelements in one cassette to influence expression.

FIG. 22: Plasmid pABExpress40 for expression of libraries ofpairing-compatible antibodies in mammalian cells. Cloning sites fordirectional insertion of antibody variable region genes are indicated.See Example 11 for details. Without the STAR40 insertion into the EcoRIsite, this plasmid is called pABExpress.

FIG. 23: Design of a hybrid light chain library for identifying apairing-compatible light chain for h4D5v8 and 2C4. The amino acidsequences used by Herceptin (trastuzumab, h4D5v8) and pertuzumab(Omnitarg, 2C4) are compared to one another, and to two designer lightchain libraries, HYB1 and HYB2 (see, Example 17 for details of thedesign). Residues identical to those of Herceptin are indicated with adash; amino acids are encoded by the single-letter consensus; X meanspositions to be targeted for diversification in a library approach.Numbers indicated for the most relevant residue positions (see, text formore details).

FIG. 24: Plasmid p2Fab-HER2 used for the identification of a light chainvariable region that is pairing-compatible with two HER2-bindingantibodies, h4D5v8, and 2C4. The black box is a schematic depiction ofthe histidine tag (six Histidines); other C-terminal-based tags are alsoindicated. S, signal sequence; rbs, ribosome binding site; AMP^(r),ampicillin resistance gene (beta-lactamase). The version of the VL ofh4D5 that is present in this vector carries two designed mutations intwo CDR residues, and a stop codon (indicated with *) in the CDR2region. By site-directed mutagenesis, the CDR2 is diversified using anoligonucleotide (designed according to approach HYB2) thatsimultaneously removes the stop codon as well as introduces diversity atthree positions of the CDR2. This plasmid directs the expression of twoantibody heavy chains (as Fd chains) and one antibody light chain, andthus allows simultaneous production, and individual detection, of twoFab fragments.

FIG. 25: Growth inhibition curves for h4D5 Fab and mixtures of 4D5* and2C4* (see, Example 17) that utilize different light chains, indicatedwith VL1 to VL7. Different concentrations of these Fabs are incubatedwith HER2-positive cells sensitive to the growth inhibitory effect ofHER2-targeting antibodies.

FIG. 26: A topology map of the annealing locations of mouse specific VHprimers and the position of required restriction sites that areintroduced by overhanging sequences at the 3′ end of primers.

FIG. 27: PCR amplification steps (Amplification, Intermediate and Siteintroduction). The location and names of the mouse VH amplificationprimers (and mixtures of primers) are indicated per step.

FIG. 28: Topology of the MV1043 vector. This vector is used for thecloning of human or murine VH fragments. O12 (IGKV1-39) is indicated asthe VL gene. Products of this vector in combination with helper phagesin E. coli cells allow the generation of phages that display Fabfragments on the surface of the phage particles as a fusion product tothe g3 protein and presence of the vector in the phage as the geneticcontent (F1 ORI).

FIG. 29: The topology of the mouse Ckappa locus downstream of theJ-segments. Both enhancers and Ckappa region are indicated. The lowerarrow indicates the region that is removed in order to silence thelocus.

FIG. 30: The topology of the mouse C-lambda locus. All three activeV-regions are indicated (Ig1-V1, V2 and V3) as are the J-segments(Igl-J1, Igl-J2, Igl-J3, Igl-J4 and the pseudo segment Igl-J3p) andconstant regions (Igl-C1, Igl-C2, Igl-C3 and Igl-C4). The regions thatare deleted in order to silence the locus are indicated by deletionmarkers. These deletions include all active V genes (1, 2 and 3) and theintergenic segment between V2 and V3.

FIG. 31: Construct topology of IGKV1-39/J-Ck with an intron located inthe leader open reading frame (ORF).

FIG. 32: Construct topology of IGLV2-14/J-Ck with an intron located inthe leader open reading frame (ORF).

FIG. 33: Construct topology of VkP-IGKV1-39/J-Ck (VkP-O12). The promoteroriginates from the IGKV1-39 gene and is placed directly in front of therequired elements for efficient transcription and translation.Intergenic sequences (including the enhancers) are derived from mice andobtained from BAC clones. The C-kappa sequence codes for the kappaconstant region of rat.

FIG. 34: Construct topology of VkP-IGLV2-14/J-Ck (VkP-2a2). The promoteroriginates from the IGKV1-39 gene and is placed directly in front of therequired elements for efficient transcription and translation.Intergenic sequences (including the enhancers) are derived from mice andobtained from BAC clones. The C-kappa sequence codes for the kappaconstant region of rat.

FIG. 35: Construct topology of VkP-IGKV1-39/J-Ck-M (VkP-012-dell) isidentical to VkP-IGKV1-39/J-Ck from FIG. 34 except that the intronenhancer region is removed.

FIG. 36: Construct topology of VkP-IGKV1-39/J-Ck-Δ2 VkP-O12-del2) isidentical to VkP-IGKV1-39/J-Ck-Δ1 from FIG. 35 except that a large pieceof the intergenic region between the Ck gene and 3′ enhancer is deleted.In addition, the 3′ enhancer is reduced in size from 809 bp to 125 bp.

FIGS. 37A-37Z: Overview of the sequences used or referred to in thisapplication: Human germline IGKV1-39/J DNA (SEQ ID NO:84); humangermline IGKV1-39/J Protein (SEQ ID NO:85); human germline IGLV2-14/JDNA (SEQ ID NO:86); human germline IGLV2-14/J Protein (SEQ ID NO:87);Rat IGCK allele a DNA (SEQ ID NO:88); Rat IGCK allele a protein (SEQ IDNO:89); IGKV1-39/J-Ck (SEQ ID NO:90); IGLV2-14/J-Ck (SEQ ID NO:91);VkP-IGKV1-39/J-Ck (SEQ ID NO:92); VkP-IGKV1-39/J-Ck-A1 (SEQ ID NO:93);VkP-IGKV1-39/J-Ck-Δ2 (SEQ ID NO:94); VkP-IGLV2-14/J-Ck (SEQ ID NO:95);pSELECT-IGKV1-39/J-Ck (SEQ ID NO:96); pSelect-IGLV2-14/J-Ck (SEQ IDNO:97); MV1043 (SEQ ID NO:98); and MV1057 (SEQ ID NO:99).

FIGS. 38A-38C: Generation of Rosa26-IgVk1-39 KI allele. (FIG. 38A)Schematic drawing of the pCAGGS-IgVK1-39 targeting vector. (FIGS.38B-1-38B-4) Nucleotide sequence of the pCAGGS-IgVK1-39 targeting vector(SEQ ID NO:100). (FIG. 38C) Targeting strategy.

FIGS. 39A-39C: (FIG. 39A) Southern blot analysis of genomic DNA of ESclones comprising an insertion of the pCAGGS-IgVK1-39 targeting vector.Genomic DNA of four independent clones was digested with Asel and probedwith 5e1 indicating the 5′-border of the targeting vector. All clonescomprise a correct insertion of the targeting vector at the 5′ end.(FIG. 39B) Southern blot analysis of genomic DNA of ES clones comprisingan insertion of the pCAGGS-IgVK1-39 targeting vector. Genomic DNA offour independent clones was digested with Mscl and probed with 3e1indicating the 3′-border of the targeting vector. All clones comprise acorrect insertion of the targeting vector at the 3′ end. (FIG. 39C)Southern blot analysis of genomic DNA of ES clones comprising aninsertion of the pCAGGS-IgVK1-39 targeting vector. Genomic DNA of fourindependent clones was digested with BamHI and probed with an internalNeo probe indicating the 5′-border of the targeting vector. All clonescomprise a correct, single insertion of the targeting vector.

FIGS. 40A-40C: Generation of Rosa26-IgV12-14 KI allele. (FIG. 40A)Schematic drawing of the pCAGGS-IgVL2-14 targeting vector. (FIGS.40B-1-40B-4) Nucleotide sequence of the pCAGGS-IgVL2-14 targeting vectorcontaining the CAGGS expression insert (SEQ ID NO:101) based on therearranged germline IGLV2-143 V lambda region (IGLV2-14/J-Ck). (FIG.40C) Targeting strategy.

FIGS. 41A-41C: EPIBASE® profile of IGKV1-39 residues 1-107. FIG. 41Adisplays the binding strength for DRB1 allotypes, while FIG. 41Cdisplays the binding strength for DRB3/4/5, DQ and DP allotypes. Thevalues in the figure represent dissociation constants (Kds) and areplotted on a logarithmic scale in the range 0.01 μM-0.1 μM (very strongbinders may have run off the plot). For medium binding peptides,qualitative values are given only, and weak and non-binders are notshown. Values are plotted on the first residue of the peptide in thetarget sequence (the peptide itself extends by another nine residues).Importantly, only the strongest binding receptor for each peptide isshown: cross-reacting allotypes with lower affinity are not visible inthis plot. The strongest binding receptor is indicated by its serotypicname. Finally, any germline-filtered peptides are plotted with a lightercolor in the epitope map (in this case, no non-self epitopes werefound). FIG. 41B shows the HLA binding promiscuity for every decamericpeptide (Y-axis: the number of HLA allotypes recognizing criticalepitopes in each of the peptides starting at the indicated residue shownon the X-axis). The promiscuity is measured as the number of allotypesout of the total of 47 for which the peptide is a critical binder. Whitecolumns refer to self-peptides, and black columns (absent here) tonon-self peptides.

FIG. 42: Epitope map of IGKV1-39 showing the presence of peptide binderspredicted in the sequence of IGKV1-39 by serotype in the 15-mer format.Each 15-mer is numbered as indicated in the top of the figure. The fullsequence of the corresponding 15-mer is listed in Table 7. Black boxesindicate the presence of one or more critical self-epitopes in the15-mer for the serotype listed on the left. Critical epitopes areoperationally defined as strong or medium DRB1 binders and strongDRB3/4/5 or DP or DQ binders.

FIGS. 43A and 43B: Constitutive knock-out (KO) of the Ig kappa locus.(FIG. 43A) Targeting strategy. (FIG. 43B) Schematic drawing of thepIgKappa targeting vector.

FIGS. 44A and 44B: Constitutive KO of the Ig lambda locus. (FIG. 44A)First step of the targeting strategy. (FIG. 44B) Second step of thetargeting strategy.

FIGS. 45A-45C: Schematic drawing of targeting vectors. (FIG. 45A)pVkP-O12 (VkP-IGKV1-39/J-Ck); (FIG. 45B) pVkP-O12-dell(VkP-IGKV1-39/J-Ck-Δ1); (FIG. 45C) pVkP-O12-del2 (VkP-IGKV1-39/J-Ck-Δ2).

FIGS. 46A-46C: Targeting strategies for insertion of transgene into theRosa26 locus by targeted transgenesis using RMCE. (FIG. 46A) VkP-012(VkP-IGKV1-39/J-Ck); (FIG. 46B) VkP-O12-del1 (VkP-IGKV1-39/J-Ck-Δ1);(FIG. 46C) VkP-O12-del2 (VkP-IGKV1-39/J-Ck-Δ2).

FIG. 47: Topology of the MV1057 vector. Replacing the indicated stufferfragment with a VH fragment yields an expression vector that can betransfected to eukaryotic cells for the production of IgG1 antibodieswith light chains containing an O12 (IGKV1-39) VL gene.

FIGS. 48A-48C: Lack of transgenic human Vk1 light chain expression innon-B cell populations of the spleen.

FIGS. 49A and 49B: Transgenic human Vk1 light chain is expressed in allB cell populations of the spleen.

FIGS. 50A and 50B: Transgenic human Vk1 light chain is expressed in B1cells of the peritoneal cavity.

FIGS. 51A-1, 51A-2, 51B-1, and 51B-2: Transgenic human Vk1 light chainis not expressed in pro- and pre-B cells but in the immature andrecirculating populations B cells in the bone marrow. (FIGS. 51A-1 and51A-2) Gating of bone marrow cells. (FIGS. 51B-1 and 51B-2) Histogramsof transgene expression with overlay from one WT control.

FIGS. 52A and 52B: Transgenic human Vk1 light chain is directlycorrelated with endogenous light chain and IgM expression in circulatingB cells in the blood.

DETAILED DESCRIPTION OF THE INVENTION

In the fight against infection, the immune system creates a cellular andhumoral response that can specifically combat the infectious agent. Thehumoral immune response is based on immunoglobulins, or antibodies,which contact antigens and mediate certain effector functions to clearthe infection ((I. M. Roit, et al. (1985)) and all references herein).In the immune system, antibodies are generated by B-lymphocytes.Antibodies consist of heavy and light chains that are assembled viainter-domain pairing and interchain disulphide bonds to form multivalentmolecules. Various isotypes of natural antibodies exist, including IgG(within humans, four subclasses, IgG1, IgG2, IgG3, IgG4), IgM, IgD, IgAand IgE. An IgG molecule contains two heavy (H) and two light (L)chains, both with a variable (V) and constant (C) regions. A typical IgGantibody comprises two heavy (H) chain variable regions (abbreviatedherein as VH), and two light (L) chain variable regions (abbreviatedherein as VL). The VH and VL regions can be further subdivided intoregions of hypervariability, termed “complementarity-determiningregions” (“CDR”), interspersed with regions that are more conserved,termed “framework regions” (FR). The extent of the framework region andCDRs has been precisely defined (see, E. A. Kabat, et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242,and C. Chothia, et al. (1987) J. Mol. Biol. 196:901-917, which areincorporated herein by reference).

In the generation of the primary immune response, the pairing of heavyand light variable region sequences of antibodies is a random process.The variable region genes are first assembled by recombining of a setrandomly picked V. (D) and J genetic elements represented in the genomeas a diverse gene pool. The recombined heavy and light variable regionsare then spliced towards their respective constant region genes and thechains expressed, assembled and secreted as immunoglobulin. In thiscombinatorial library, in principle every heavy chain can pair withevery light chain, to create a vast repertoire of different antigenspecificities, with diversity derived from the rearrangement process(which also introduces further diversity at some of the segmentjunctions) and from the combinatorial assembly of the heavy and lightchain variable regions. In principle, B-cells produce only one antibodyspecificity, encoded by one antibody heavy and one antibody light chainsequence. The immune system selects via an efficient antigen-selectionprocess those antibodies that can bind to a given antigen, inparticular, when the antigen is foreign and part of a pathogen.

In natural immunoglobulins, the light chain which consists of twodomains, is paired to the heavy chain, which consists of at least fourdomains and a hinge region: non-covalent interactions occur between VHand VL, and between CH1 and CL; between the latter a disulphide bridgeprovides a covalent linkage between heavy and light chains. Furthermore,the heavy chains are found paired to one another, i.e., in the IgGformat, and sometimes further associate with additional elements such asJ-chains (e.g., in the IgM format). A strong non-covalent interactionoccurs between the CL and CH1 domains, a frequently weaker interactionis present between VL and VH. The heavy chains are paired viainteractions in the hinge region (often covalently associated via one ormore disulphide bridges) and between the CH2 and CH3 domains. Bysequencing large pools of antibody variable genes from isolated

B-cell and comparing the frequency of the pairings of VH and VLsegments, it was confirmed that this pairing between VH and VL regionsis on average a random process. However, since the variable regions aregenetically diverse and some of this diversity at the amino acid levelis structurally situated at the predicted interface region between thetwo domains, the pairing of one given VH to another VL is not any morerandom. For example, pairing of a given VH with another VL than themolecule was initially selected with, may lead to loss of affinity ofbinding for the antigen, but may also lead to a reduced pairingefficiency. Within one B-cell, typically and normally only one light andone heavy chain is expressed, but in the few instances that other lightor heavy chains are expressed (such as in two fused B-cells), mispairingbetween the chains will occur, and antigen binding is lost in thisfraction of the antibody preparation. For example, in the past, theexpression of multiple antibody variable domains, as in quadromas orcells transfected with multiple heavy and/or light chain genes,typically yields a large fraction of pairings of variable regions thatare not functional. In order to build bispecific antibodies, the pairingof different antibody heavy and light chains when expressed in the samecell was investigated intensively. From studies of the pairing inantibodies derived from hybrid hybridomas made by fusing twoantibody-producing hybridomas, the pairing was shown to be based on arandom association of light and heavy chains with some cases where acertain level of preferential pairing was seen, but not enough toprevent mispairing to occur.

Described herein are a variety of methods to select antibodies withoptimal pairing behavior of antibody chains With such methods,compositions of multiple antibodies with different binding specificitiescan be made.

1. Antibodies with Pairing-Compatible Variable Regions

a. Summary

Herein, disclosed are methods and means for obtaining antibodies withpairing-compatible variable regions. The presence of such variableregions facilitate the predictability and functionality of the resultingpairing between the antibody variable regions. Two antibodies containpairing-compatible variable regions when the pairing of the variableregions in a mixture of all variable regions combined, occurs in suchmanner that predominantly functional binding sites arise as a result ofthe pairing. Two antibodies have pairing-compatible variable regionswhen, for example, the variable light chain domains of both antibodiescan be exchanged by the one of the other antibody, without drasticallyaltering the antigen-binding affinity of the two antibodies. Anotherexample of when antibodies have pairing-compatible variable regions, iswhen they share an identical or closely related variable region. In thatcase, pairing of the two partner domains to this shared region will leadto the formation of functional binding sites.

Methods for the identification of antibodies that havepairing-compatible variable regions are described. In the simplest form,pairing-compatible variable regions in sets of antibodies are identifiedby virtue of the sequence identity of the V-regions. In anotherapproach, pairing compatible variable regions are identified byempirical exchange of V-genes or V-gene fragments between givenantibodies, and testing antigen binding. In another approach, antibodieswith a high likelihood of containing pairing-compatible variable regionscan be enriched from antibody repertoires by combinations of selectionsand re-shuffling. Using appropriate selection strategies, antibodypairing may be selected to become promiscuous or exclusive in thecontext of the desired multiple antibody variable genes. A method isalso described for providing a given antibody with pairing-compatiblevariable sequencing, using various mutagenesis and selectiontechnologies. In another approach, antibodies with pairing-compatiblevariable regions are selected from synthetic antibody libraries with ahigh probability of identifying antibodies with such elements (forexample, from a library with only one variegated variable domain).Further, antibodies with pairing-compatible variable regions are createdby first selecting an antigen-specific single-domain antibody, and thenproviding this with a second domain that will pair with the first one toform a two-domain molecule.

Pairing-compatible variable regions can be identified in order toreplace sequences in an antibody by the equivalent sequences of anotherantibody that are thought to mediate more favorable characteristics. Thetransfer of pairing-compatible variable regions between antibodies canbe used to alter the pairing capability and pairing strength of theantibody chains, but it can also be envisaged to alter theimmunogenicity, idiotype and expression yield of antibodies. Antibodiesbearing such elements are also highly suitable for making pharmaceuticalcompositions of antibodies with multiple binding sites, for example, formaking mixtures of antibodies containing such elements, by co-expressionin the same host cell. In particular, when the variable regions share afull variable domain (such as the light chain), co-expression will yieldfunctional binding sites only. Antibodies with pairing-compatiblevariable regions are suitable for the creation of mixtures ofantibodies, in which the antibodies are either solely monospecific, orbispecific, or a mixture of mono- and bispecific antibodies, or even,depending on the choice of isotypes with more than two binding sites(e.g., sIgA, IgM), combinations of multiple specificities within thesame antibody molecule. Such approaches provide a means to have in thesame pharmaceutical preparation antibodies with multiple specificities,and, if required, combinations of specificities within the samemolecule.

b. Sources of Antibodies

Suitable antibodies can be derived from a variety of sources, includingmonoclonal antibodies, phage antibodies, antibodies from transgenicanimals, etc. Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies using a hybridoma method firstdescribed by Kohler and Milstein, Nature 256:495 (1975) or may be madeby recombinant DNA methods. In the hybridoma method, a mouse or otherappropriate host animal, is immunized to elicit lymphocytes that arecapable of producing antibodies that will specifically bind to theantigen used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes are fused with myeloma cells using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Antibodies can also be isolated from transgenic animals thatharbor human immunoglobulin genes.

Antibodies or antibody fragments can also be isolated usingdisplay-based antibody library technology, wherein antibody fragmentsare selected by exposing a library of such antibodies displayed on thesurface of phage, yeast or other host cell, to the antigen of interest,and isolating those antibody fragments which bind to the antigenpreparation. A display library is a collection of entities; each entityincludes an accessible polypeptide component and a recoverable componentthat encodes or identifies the peptide component. Many antibodyfragments have been displayed on the surface of entities that carry thegenetic material encoding the antibody fragment inside the entity, suchas bacteriophages. This format is termed “phage display.” Phage displayis described, for example, in Ladner et al., U.S. Pat. No. 5,223,409;Smith (1985) Science 228:1315-1317. Other display formats utilizepeptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can begenerated by the in vitro translation of mRNA that includes a covalentlyattached puromycin group, e.g., as described in Roberts and Szostak(1997) Proc. Natl. Acad. Sci. U.S.A. 94:12297-12302, and U.S. Pat. No.6,207,446. The mRNA can then be reverse transcribed into DNA andcross-linked to the polypeptide. In still another display format thelibrary is a cell-display library. Proteins are displayed on the surfaceof a cell, e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryoticcells include E. coli cells, B. subtilis cells, spores, exemplaryeukaryotic cells include yeast such as Saccharomyces cerevisiae,Hansenula polymorpha, Pichia pastoris, Kluyveromyces lactis, insectcells, and mammalian cells. Methods for the display of antibodyfragments and the construction of antibody libraries in a variety offormats are well described in the literature and known to those skilledin the art.

c. Identifying Pairing-Compatible Elements in Panels of Antigen-ReactiveAntibodies

Antibodies with pairing-compatible variable region sequences and,therefore, suitable pairing behavior of variable regions, are identifiedby a variety of methods that are disclosed within this document. In afirst approach, antibodies with pairing-compatible variable regions areselected from panels of antigen-specific antibodies (in which theantigen can be one defined target antigen but also a collection ofdifferent antigens, and the panel contains at least two antibodies), asfollows. The sequences of heavy and light variable regions aredetermined and inspected to find clones with identical or highly similarlight or heavy chain variable domains. If the amino acid sequence ofpart of or the complete variable region is identical for two antibodies,the two given antibodies have a pairing-compatible variable region.

In another approach, pairing-compatible variable regions are identifiedin amino acid sequences that appear related yet have amino aciddifferences: for example, if there are differences in the amino acidsequence but the same or related germ line segment is used, or whenhighly similar CDR regions are used, or if similar canonical folds insome CDR regions are found yet different germ line segments are used,the variable regions may still comprise pairing-compatible variableregions. This is confirmed by swapping the variable region(s) betweenthe antibodies in the panel, and measuring antigen binding of the newpairs. Experimentally light and heavy chains or parts thereof can beexchanged by recombinant DNA methods such as restriction enzyme-basedDNA cloning, oligonucleotide-based mutagenesis, gene synthesis andPCR-mediated mutagenesis, methods which are widely available in the art.Binding assays that can be used are well established in the art andknown to those skilled in the art; some are described below. This methodmay identify cases in which both variable regions can be exchangedbetween two antibodies, such as two related light chains that can beswapped with no or an acceptable effect on the affinity. It can alsoidentify cases in which only one of the variable regions of the twoantibodies can tolerate the exchange, for example, one light chain thatfunctionally pairs with one of two heavy chains only, while the otherlight chain can functionally pair with both heavy chains. In that casethe latter light chain can be used to replace the former non-matchingone and, thus, create two antibodies with pairing-compatible variableregions. Functional pairing means that the variable region pairing hasideally no effect on antigen-binding affinity or specificity, butallowable may also be a <10-fold reduction in affinity, and at the mosta 100-fold reduction in affinity, or any improvement of affinity.

In another embodiment, pairing-compatible variable regions areidentified in panels of antibodies without knowing or using the sequenceof the variable regions of the antibodies. First a collection ofantibody variants is created in which all variable regions are combinedwith the other partner variable regions of the antibodies in the panel.Then, the effect on antigen binding is established empirically, toidentify those antibodies with can functionally pair to the variableregions of the other antibodies in the panel (FIG. 2). This methodidentifies pairing-compatible variable regions that are not immediatelyidentified by sequence comparison. Instead of using the partner variableregions derived of the antibodies in the panel, also other partnervariable regions can be used. For example, the heavy chain variableregion of each of the antibodies in the panel is combined with a set ofchosen light chain variable regions, for example, consisting of mainlygerm line encoded segments representative of one or more of the lightchain kappa or lambda gene families Pairing-compatible variable regionsare then identified by screening the combinations for antigen bindingand scoring whether one common variable region provides antigen bindingfor the desired set of antibodies in the panel. These methods can bebased on assessment of antigen binding of individual combination of thevariable region genes, thus co-expression of two variable regions in thedesired antibody format, or of antigen binding of multiple combinationsof variable regions derived from co-expression in the same host cell.For example, two antibody heavy chain variable regions can be expressedinside the same host cell as Fd chain, and co-expressed with one lightchain, and antigen binding for both antibody binding sites assessed.Further, by differentially tagging the two heavy chains, for example,with epitope tags such as tags derived from c-myc, VSV, HA, etc., thepairing of the two H-L combinations can be followed. Such an approach issuitable for finding pairing-compatible variable regions if a limitednumber of starting antibodies is available and allows the screening oflarge collections of partner variable regions.

Examples of pairing-compatible variable regions are V-regions based uponhighly homologous germ line segments, or V-regions that differ bychanges in the amino acid sequence (e.g., with somatic or othermutations, minor deletions, additions, substitutions). In such case, theeffect of the exchange of the homologous region in the first antibodymay differ from the effect seen with the exchange of the homologousregion in the second antibody; e.g., there are cases where the affinityis changed to an allowable level for only one of the two antibodies, andcases where this occurs for both antibodies. In one embodiment, thepairing-compatible variable region comprises the light chain variableregion or part of the light chain variable region. In anotherembodiment, the pairing-compatible variable regions comprise the heavychain variable region or part of the heavy chain variable region.

Another embodiment of an approach to identify pairing-compatiblevariable regions in a panel of antibodies is the following. First thevariable region of each of the antibodies is co-expressed with a partnervariable region derived from the other antibodies in the panel, and ascreen carried out that will detect the presence of intact antibody(thus, not antigen binding). The formation of intact antibody indicatespairing between the two variable regions; if no intact antibody isretrieved, this will indicate that the two variable regions are notpairing inside the host cell. The screening can be used to identifyantibodies that display variable regions that cannot pair with oneanother in the chosen antibody format, i.e., as Fab fragments expressedin E. coli or as IgG molecules expressed in eukaryotic cells. Whenco-expressing the four variable region genes, only the cognateinteractions occur, and the variable region genes arepairing-compatible.

d. Antibodies with Pairing-Compatible Variable Regions from AntibodyLibraries

In certain embodiments, antibodies with pairing-compatible variableregions are selected from synthetic antibody libraries with a highprobability of identifying antibodies with such elements. Syntheticantibody libraries are collections of antibodies that have beensynthetically diversified (e.g., using site-directed mutagenesis orPCR-based gene synthesis using mutagenized oligonucleotides) in definedregions/locations within their variable regions. In one embodiment, thedesign of the diversity introduced into the primary antibody repertoireis such that at least a portion of a variable region and, for example, acomplete variable region is not diversified, while the remaining areacontains the diversity (examples in FIGS. 3, 4(a), 4(c) and 4(d)).Examples of such libraries are libraries based on human variable regiongenes, for example, a set of 49 different heavy chain genes withdiversity introduced in the VH-CDR3, all combined with a single lightchain (H. R. Hoogenboom, et al. (1992) J. Mol. Biol. 227:381-388).Antibodies selected from such repertoires will contain by designpairing-compatible variable regions. Such repertoires can be created byrecombinant DNA methods and displayed on the surface of phage, cells,spores, ribosomes, or can be created in transgenic mice carrying onlypartial diversity in the V-gene composition. Synthetic diversity can beintroduced in all CDR residues, in a subset of CDR residues, i.e., thosewith significant solvent exposure, and can be designed to encode all ora subset of amino acids, i.e., those that are commonly observed innatural antibody CDRs. An example of such tailored antibody library,with a single heavy chain variable domain scaffold and a fixed lightchain variable domain, and with a limited number of heavy chain CDRresidues variegated with a limited number of encoding amino acids isdescribed in J. Mol. Biol. 338:299-310 and in WO 03/102157A2.Alternatively, to libraries with synthetic diversity in one variableregion, also libraries with natural diversity, or combinations ofnatural and synthetic diversity (e.g., synthetic diversity in CDR1 andCDR2 and natural diversity in CDR3) in one variable region may be used.

In one embodiment, antibodies with pairing-compatible variable regionsare obtained by first selecting an antigen-specific single-domainantibody, and then providing this with a second domain that will pairwith the first one to form a two-domain molecule (examples in FIG. 4,columns (b) and (e)). Single-domain antibodies may be isolated from invitro display repertoires made from single-domain repertoire of certainhuman variable region fragments, such as human VH or human VLrepertoires. In another embodiment, single domain antibodies areisolated from non-immunized, immunized or synthetic VHH repertoires,based on antibody heavy chain domains naturally devoid of light chains(e.g., camel, lama or some shark antibodies). Single-domain VH-basedantibodies with antigen-binding activity can be combined via recombinantDNA technology with a single, a small repertoire, a chosen collection ora large repertoire of light chains, for example of human nature.Antigen-binding variants of single-domains now forced to contain apaired light chain, may be isolated using display technology based orequivalent methods. In another embodiment, single-domain VL-basedantibodies with antigen-binding activity are combined via recombinantDNA technology with a single, a small repertoire, a chosen collection ora large repertoire of heavy chains, for example of human nature.Antigen-binding variants of single-domains now forced to contain apaired heavy chain, may be isolated using display technology-based orequivalent methods. In the embodiments of FIG. 5, the variants derivedfrom the same route of isolation will always share a variable regionsequence, thus will be able to provide functional pairing when broughtinto the context of pairing multiple variable regions.

If at least a portion of a variable region and a complete variableregion is not diversified, while the rest of the variable region(s)contain the diversity, the selected antigen-binding antibodies comingfrom such repertoires will contain by design pairing-compatible variableregions. In many of the approaches in the literature used for buildinghigh affinity antibodies from synthetic antibody libraries, diversity inthe initial library is built up throughout the antibody variable regiongenes and, in particular, in most of the six CDRs. Depending on thegenetic make-up of these libraries, there will be a higher or lowerprobability of identifying antibodies with pairing-compatible variableregions. Libraries can be designed to fit specifically this newapplication, by introducing diversity in one variable region only, andnot further diversifying the shared variable region, even in furtheraffinity maturation processes. Libraries may be used in which thediversity is restricted to the three CDRs in one chain Thepartner-variable region may then be, for example, one or a small set ofgerm line gene-encoded regions without any further diversity. In theprimary library or follow-up libraries, diversity can be introduced inthose areas of the antibody V-regions that are less likely to interactwith the partner chain, so as to increase the chances of findingantigen-binding antibodies with high affinity, yet well pairing variableregions.

Antibodies with a high likelihood of containing pairing-compatiblevariable regions can also be enriched from antibody repertoires notbiased in their genetic make-up, by combinations of selections andre-shuffling of, for example, the complete V-region of a givenpopulation or clone (exemplified in FIG. 5). This will enrich for thoseantigen-specific antibodies with a high likelihood of containingpairing-compatible variable regions, for example, because they aretolerant in their pairing with the shuffled region yet retainantigen-binding, or because the shuffled region is less likely tocontribute to antigen binding. For example, an antibody Fab library isfirst enriched on antigen, and the selected heavy chains obtained afterone or more rounds of selection are then recombined with the selected orunselected light chain repertoire (dashed lines in FIG. 5), and selectedagain on antigen (FIG. 5, step 4). In this way the selected antibodyvariable heavy chain domains will have the propensity to bind to theantigen relatively independently of the light chain to which it ispaired. Antibodies to a first and second antigen can be identified byusing the above-described selection and re-shuffling experiment,followed by a screening as before, to detect antigen binding of theselected heavy chains in combination with a collection of light chainsOne may then identify those antibodies that bind either the first or thesecond antigen relatively independently of the light chain, or in thepresent of a related light chain family member. Due to the dominance ofthe heavy chain in antigen binding in these antibodies, many of thelight chains are likely to functionally pair with the multiple heavychain variable regions. Co-expression of antibodies with apairing-tolerant variable region that is mediating antigen binding (suchas the VH), and in which the partner domain is not involved or notimportant for antigen binding (such as the VL), will similarly lead tothe formation of mainly or only functional binding sites.

In another embodiment, described is a method to obtain antibodies withheavy and light variable regions that preferentially or in the bestcase, exclusively, pair to one another and not to the respective lightand heavy variable regions of one or more other antibodies, for example,those that are co-expressed in the same host. Such selection can be doneby display methodology, but also using an intracellular selection routethat relies on co-expression of antibody light and Fd chains in the samecell, allowing competition between the chains, and rescue of theintended combination via phage display or any other suitable route. Thepreferential or ideally exclusive pairing that is encountered infaithful antibodies will aid in the formation of mainly or onlyfunctional binding sites when such antibodies are co-expressed. Thismethod essentially allows a high level of functional antibody bindingsites to form even when variable region genes are used that have verydistinct compositions.

A method for identifying antibodies with desired pairing behavior basedon competition selection is described here. Antibodies are selected froma library of antibody fragments, by carrying out a selection directly ina host cell that co-expresses different antibodies. For example, whenapplied to using bacteriophage libraries, this concept is the following:bacteria are provided with a phage or phagemid genome that carries thegenes encoding a Fab fragment in such manner that upon expression, oneof the chains will be anchored to a phage particle. In the same hostcell, other antibody light and/or heavy chain Fd fragments areco-expressed, for example, the Fab genes encoding a given antibody, orany set of multiple antibodies. For example, consider co-expression oftwo Fabs in the same cell, one of which is anchored via its heavy chain(Fd fragment, essentially VH-CH1) to the phage coat protein. As aconsequence of this co-expression, competition occurs inside the samecell (in this case in the periplasm) between the two light chains forthe pairing to the phage-anchored Fd chain Further, the soluble heavychain of the competing Fab will be able to pair with both light chainspresent in the same cell. In this system, phage particles with antigenbinding activity will occur with different types of pairings. First, ifthe correct light chain will pair with its partner heavy chain on thephage only (exclusive pairing), and second, if the heavy chain on thephage surface is dominant in antigen binding and tolerant for pairing,yielding antigen binding virtually irrespective of which light chain itpairs with. Functionally such antibody pairs will behave in the samemanner In the case of the first situation, the lesser interactionsbetween the partners of the two respective antibody pairs, the higherthe proportion of functional Fab on phage.

The method described can be further biased towards antibodies with anexclusive pairing, by providing tags on the chains and enriching ordepleting for particular combinations (e.g., depleting for those phagethat carry the competitor light chains via a unique tag present on thesechains) This method when applied to the isolation of antibodies via theselection of a phage library of Fabs, will yield a high frequency ofantibodies that will have an appropriate pairing behavior and highfunctional yield when produced as mixture by co-expression. The use ofcompetition-selection to bias selected antibodies towards beingco-expression compatible, may also be applied to other display libraries(e.g., yeast display libraries), and to in vitro library systems basedon ribosome display or mRNA display (Puromycin system), with methods ofscreening or selection of antibodies that recognize antigen asextensively described in the art. Further, the described method ofcompetition-selection of antibody fragments for improved pairing (orantigen-selection and compatible pairing) using phage display can bereadily translated into an intracellular (periplasmic) selection systembased on protein-or enzyme complementation. In such approaches,fragmented, complementary or self-inhibitory enzymes are used to drivethe selection of interacting molecules that are fused to the componentsof the selection system. Only when there is an interaction of a minimalstrength will the protein or enzyme become activated, and underappropriate selection conditions, will the cells survive. Such methodshave, for example, been described for the enzymes beta-lactamase andDHFR, with its applications in the selection of antibodies or expressedcDNA fragments that display a particular binding behavior. For example,competitive selection has been described for the affinity maturation ofantibodies in the TACZYME system from Kalobios Inc. Herein, it is notthe antigen binding but the pairing strength that can be made theselective force for a given population of antibodies presented in suchsystem.

In some embodiments, the method is used to identify new antibodies fromphage libraries that show pairing-compatible variable regions with anexisting antibody that has given variable region sequences. The antibodywith the known antigen specificity is cloned for co-expression as Fabfragment in host cell that collectively express a phage display libraryof human Fab antibodies. This can be done by providing the Fabexpression cassette onto a plasmid that is compatible with the presenceof a phage or phagemid genome, such as the pBR322-based plasmid. Hostcells harboring this plasmid are then infected with the phage particlesencoding a library of human Fabs cloned into, for example, a phagemidvector such as pUC119, or a phage vector such as fd-tet-DOG1. While thecompeting Fab fragment is expressed, new phage particles are harvested(after helper phage infection if appropriate) from this culture. Theseparticles are used for selection on antigen, and the resulting phagereinfected into cells harboring the competitor Fab fragment. After a fewiterative rounds, the phage Fabs are screened for antigen binding in abinding assay; the pairing behavior between the reactive Fabs and thevariable regions of the competing Fab can be further tested byco-expression and binding assays. One example of a format for thisselection is the Fab format and not the scFv format, mainly because formost applications whole IgG-type antibodies will need to be establishedthat have interactions between the chains that harbor the variableregions that mimic those seen in the Fab format. FIG. 6 depicts anexample of how this method works for two Fabs competing with antibodiesin a phage library.

This method requires some optimization steps, e.g., the use of aCH1-mutant with reduced affinity for its CL, and Fabs that do notdisplay an intermolecular disulphide bridge such that the pairing willremain noncovalent. Residues positioned at the CH1-CL interface regionmay be mutated such that affinity between these two domains is reduced,for example, 10-fold or 100-fold, and as a result in the Fab format thepairing of the variable domains will become more dominant in driving thetwo chains together. Antibodies selected from such mutated Fablibraries, or from Fv libraries in which there is no covalentassociation between the two variable regions, may be biased towardshaving a preferential pairing behavior.

In a further embodiment, described is the creation of antibody librariesin which provisions are made to mediate unique pairing between the heavyand light chains, such that they are unlikely to interact withantibodies derived from a “regular” or non-purposely biased composition.An example of such provision is a knobs-into-holes engineered CH3-CH3pair, in which one domain is provided with an amino acid with a large,bulky side chain (e.g., a tyrosine; the knob) that pokes out into theinterface region, while the other domain at the equivalent structuralposition, carries one or more mutations (e.g., three) to create a holeinto which the “knob” will fit. Examples of such engineered domaininterfaces have also been published for variable regions (Zhu et al.(1997) Protein Science 6:781-788). It was shown that the effects ofdomain interface mutants are context (antibody) dependent, whichprovides also an opportunity to engineer the variable region domaininteractions in an antibody-specific manner, in such way that whenmultiple antibody variable gene pairs are allowed to pair, mainly oronly the cognate pairings are retrieved. Alternatively, installing adisulphide bond between the domains may mediate a preferential pairing.Alternatively, charge replacements are introduced in the frameworkregions, or combinations of these with sterically complementarymutations, to disfavor mispairing with one, and/or more favorablepairing with the other partner variable region. Selection systems forsuch mutant libraries have been described earlier, and include theselection of the domain libraries on antigen via phage display of thepaired variable regions (in scFv or Fab or, IgG format), or ribosomedisplay of the scFv fragments, or selections based on the interactionitself instead of that with antigen. An example of the latter isdescribed for selecting heterodimers of the immunoglobulin gamma-1 CH3domain (Atwell et al. (1997) J. Mol. Biol. 270:26-35), which isapplicable as follows: on of the two variable regions that should orshould not interact (depending on what one would like to select for,repulsion or attraction/pairing) is displayed on phage (for example asVLCL or as VHCH1 chain), while the other is genetically tagged andproduced in solution (for example as VHCH1 or as VLCL). The interactionbetween the two variable regions can than be selected for, usingstandard phage selection protocols and anti-tag reagents. Co-expressionwith a pair of non-tagged competitor variable regions as describedearlier can be used to drive the selection towards variable region pairsthat exclusively pair with one another.

In another embodiment of selecting binding sites with appropriatepairing behavior, described here are the use of antibodies derived fromVH-VH libraries on the one hand and VL-VL libraries on the other; or theuse of chimeric libraries in which elements (one or more CDR regions)are swapped between VH and VL. In another embodiment, comprised is thecreation of two antibody libraries with such provisions made to mediateunique pairing between the heavy and light chains, such that whenantibodies from these libraries are co-expressed, they will likelypreferentially pair with the right partner.

Cited libraries of antibodies can take various forms. As a source ofantibodies, a naive human library may be used, such as the antibodylibraries described by Griffiths (A. D. Griffiths, et al. (1993) EMBO J.12:725-734), Vaughan (T. J. Vaughan, et al. (1996) Nat. Biotechnol.14:309-314), or de Haard (H. J. de Haard, et al. (1999) J. Biol. Chem.274:18218-18230). Both heavy and light chains in these libraries arederived from the repertoires of rearranged V-genes derived from the mRNAof peripheral blood lymphocytes (PBLs) from unimmunized humans and are,therefore, highly diverse. Alternatively, as a source of antibodies animmunized host or patient with biased humoral response (e.g., patientswith infections, autoimmune diseases, etc.) is used. In immune librariesmade from a hapten-immunized animal, it was shown that many of theclones were promiscuous and allowed pairing of the originally selectedheavy and light chains with partner chains derived from other selectedclones. Thus, antibodies with pairing-compatible variable regions may bemore frequent in such immune libraries than in non-immune libraries.

Cited selection and screening technologies of recombinant antibodies andtheir fragments are well established in the field. Antigen-specificpolypeptides can be identified from display libraries by directscreening of the library, or can be first selected on antigen toincrease the percentage of antigen-reactive clones. The selectionprocess may be accomplished by a variety of techniques well known in theart, including by using the antigen bound to a surface (e.g., a plasticsurface, as in panning), or by using the antigen bound to a solid phaseparticle which can be isolated on the basis of the properties of thebeads (e.g., colored latex beads or magnetic particles), or by cellsorting, especially fluorescence-activated cell sorting (FACS). As willbe apparent to one of skill in the art, the antigen-specific affinityreagent may be bound directly or indirectly (e.g., via a secondaryantibody) to the dye, substrate, or particle. Selection procedures havebeen extensively described in the literature (see, e.g., Hoogenboom(1997) Trends Biotechnol. 15:62-70). Other publications describe theproduction of high affinity (nanomolar range) human antibodies from verylarge collections of antibodies, and the affinity maturation of theseantibodies by chain shuffling or other approaches (reviewed in, e.g., H.R. Hoogenboom, et al. (2000) Immunol. Today 21:371-378). Binding ofantibodies to their respective antigens may be carried out usingantibody-based assay techniques, such as ELISA techniques, Westernblotting, immunohistochemistry, Surface Plasmon Resonance (SPR)analysis, affinity chromatography and the like, according to methodsknown to those skilled in the art (see, for example, Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory Press). These techniques are viable alternatives tothe traditional hybridoma techniques for isolation of “monoclonal”antibodies (especially when human antibodies are required), which areencompassed herein.

The following describes possible embodiments of exemplary assays forbinding assays: ELISA. Polypeptides encoded by a display library canalso be screened for a binding property using an ELISA assay. Forexample, each polypeptide is contacted to a microtiter plate whosebottom surface has been coated with the target, e.g., a limiting amountof the target. The plate is washed with buffer to removenon-specifically bound polypeptides. Then the amount of the polypeptidebound to the plate is determined by probing the plate with an antibodythat can recognize the polypeptide, e.g., a tag or constant portion ofthe polypeptide. The antibody is linked to an enzyme such as alkalinephosphatase, which produces a colorimetric product when appropriatesubstrates are provided. The polypeptide can be purified from cells orassayed in a display library format, e.g., as a fusion to a filamentousbacteriophage coat. In another version of the ELISA assay, eachpolypeptide of a library is used to coat a different well of amicrotiter plate. The ELISA then proceeds using a constant targetmolecule to query each well.

Surface Plasmon Resonance (SPR). The binding interaction of a moleculeisolated from library of diversity strands with a target can be analyzedusing SPR. For example, after sequencing of a display library memberpresent in a sample, and optionally verified, e.g., by ELISA, thedisplayed polypeptide can be produced in quantity and assayed forbinding the target using SPR. SPR or Biomolecular Interaction Analysis(BIA) detects biospecific interactions in real time, without labelingany of the interactants. Changes in the mass at the binding surface(indicative of a binding event) of the BIA chip result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance). The changes in the refractivity generatea detectable signal, which are measured as an indication of real-timereactions between biological molecules. Methods for using SPR aredescribed, for example, in U.S. Pat. No. 5,641,640; Raether (1988)Surface Plasmons, Springer Verlag; Sjolander and Urbaniczky (1991) Anal.Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705 and on-line resources provide by BIAcore International AB(Uppsala, Sweden). Information from SPR can be used to provide anaccurate and quantitative measure of the equilibrium dissociationconstant (K_(d)), and kinetic parameters, including k_(on) and k_(off),for the binding of a biomolecule to a target. Such data can be used tocompare different biomolecules. For example, proteins encoded by nucleicacid selected from a library of diversity strands can be compared toidentify individuals that have high affinity for the target or that havea slow k_(off). This information can also be used to developstructure-activity relationships (SAR). For example, the kinetic andequilibrium binding parameters of matured versions of a parent proteincan be compared to the parameters of the parent protein. Variant aminoacids at given positions can be identified that correlate withparticular binding parameters, e.g., high affinity and slow k_(off).This information can be combined with structural modeling (e.g., usinghomology modeling, energy minimization, or structure determination bycrystallography or NMR). As a result, an understanding of the physicalinteraction between the protein and its target can be formulated andused to guide other design processes.

Homogeneous Binding Assays. The binding interaction of candidatepolypeptide with a target can be analyzed using a homogenous assay,i.e., after all components of the assay are added, additional fluidmanipulations are not required. For example, fluorescence resonanceenergy transfer (FRET) can be used as a homogenous assay (see, forexample, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos etal., U.S. Pat. No. 4,868,103). Another example of a homogenous assay isAlpha Screen (Packard Bioscience, Meriden Conn.). Alpha Screen uses twolabeled beads. One bead generates singlet oxygen when excited by alaser. The other bead generates a light signal when singlet oxygendiffuses from the first bead and collides with it. The signal is onlygenerated when the two beads are in proximity One bead can be attachedto the display library member, the other to the target. Signals aremeasured to determine the extent of binding. The homogenous assays canbe performed while the candidate polypeptide is attached to the displaylibrary vehicle, e.g., a bacteriophage.

Automated screening. The methods and compositions provided herein arealso suitable for automated screening of diversity libraries for findingclones with likely pairing-compatible variable regions. For example, adisplay library of Fabs or scFvs can be screened for members that bindto a target molecule. The library can be screened directly or firstselected on antigen once or several times. Binders from a first round ofscreening can be amplified and rescreened, one or more times. Bindersfrom the second or subsequent rounds are individually isolated, e.g., ina multi-well plate. Each individual binder can then be assayed forbinding to the target molecule, e.g., using ELISA, a homogenous bindingassay, or a protein array. These assays of individual clones can beautomated using robotics. Sequences of the selected clones can bedetermined using robots and oligonucleotide primers that allow to readthe variable region sequences of the selected clones. Results of theassay and the sequences can be stored in a computer system and evaluatedby eye or by using software, e.g., to identify clones which meetparticular parameters (e.g., for binding affinity and/or specificity,and for sequence homology).

e. Forcing Appropriate Pairing of Antibody Variable Regions via Mutationand Selection

There are instances where antibodies with given variable regionsequences, antigen specificity and affinity are available, but where nopairing behavior can be achieved with the existing sequences. Some ofthe methods mentioned earlier can be applied to solve this, inparticular, the screening of a combinatorial panel of variable regionpairs to find fortuitously compatible pairs, or the selection of newantibodies that do have the desirable pairing behavior, for example,using competition selection with one of the antibodies of definedspecificity. In those instances where this is not a desirable option andthe existing antibodies are used, the following methods may be used tocreate pairing-compatible variable regions for the set of antibodies tobe produced as an OLIGOCLONICS® mixture.

First of all the pairing can be biased by using single-chain Fv variantsof the antibodies. The provision of a linker between heavy and lightchain variable region will increase the chance that the two domains willpair with one another, instead of pairing with unlinked molecules orwith other single chain Fv molecules of the same or differentspecificity present in the same cell. If such molecules are fused to Fcregions and co-expressed in the same host cell, the result is a mixtureof scFv-Fc molecules which are paired via the heavy chain Fc region,forming monovalent and bispecific molecules. There is also analternative solution that does not rely on pairing in the scFv format.With a set of, for example, three given antibodies, an antibody mixtureconsisting essentially of IgG-formatted molecules can be made by makingthe variable region genes compatible with one another. First thesequence of the antibody light chains is determined, and the chain thatis the most common to the sequence of the two other light chain variableregions, or the closest to its germ line amino acid sequence identified.For the two antibodies that carry the different light chain, a libraryof heavy chains is created that is diverse in the CDRs including theCDR3 that produces a substantial fraction of the interactions betweenheavy and light variable region sequences. These heavy chains arecombined with the chosen, non-mutated light chain in a format thatprovides expression and screening, or display and selectioncapabilities. In such manner, the two remaining antibodies are forced toaccept the new light chain, which could affect pairing and affinity; theprovision of mutations in the heavy chains and the selection (eitherseparately as scFv or Fab fragments, or as Fab in competition with theiroriginal light chain in a method described above for competitionselection), will enrich for variants that have corrected a possibledeficiency in pairing efficiency and/or affinity loss.

f. Antibodies with Pairing-Compatible Variable Regions from TransgenicMice

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Transfer of the human germ-line immunoglobulin gene array in mutant micethat carry a homozygous deletion of the antibody heavy chain joiningregion (JH) gene and, therefore, do not anymore produce murineantibodies, results in the production of human antibodies upon antigenchallenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A.90:2551-255 (1993); Jakobovits et al., Nature 362:255-258 (1993).Antibodies with pairing-compatible variable regions may be identifiedfrom panels of antibodies made in these animals, or from such antibodiesand antibodies derived from other methods. It is envisaged thatantibodies with pairing-compatible variable regions may be identifiedeven more readily in transgenic mice carrying only the heavy or only thelight chain locus, and only a single or a limited set of chosen partnerchains; in that case immunization would lead to the generation ofantibodies which all carry a compatible common chain Antibodies withpairing-compatible variable regions are then identified using themethods described herein. The efficiency with which such antibodies canbe identified can be further increased by reducing the extent of somatichypermutation of the partner chain or chains. This can, for example, bedone by removing regulatory sequences surrounding the variable regions,or by mutating the variable region codons such that the gene becomes aless likely substrate for the cellular hypermutation machinery, or byharvesting the B-cells earlier after immunizations.

One further approach is to combine the heavy chains of the threeantibodies with a repertoire of highly diverse light chains, and screenthe pairings, if necessary after selection on antigen, for light chainsthat maintain functional pairing (and antigen binding) and share acommon sequence. This can be readily carried out using automatedfacilities for high throughput ELISA screening and sequencing, aspresented earlier.

g. Uses of Antibodies with Pairing-Compatible Variable Regions

Antibodies with pairing-compatible variable regions have manyapplications. It is disclosed herein that the preparation of a desiredfunctional antibody mixture is feasible when the composition of thevariable heavy or light chains of the various antibodies is carefullyselected to contain antibody variable regions that carrypairing-compatible variable regions such that the pairing of theantibody variable regions yield predominantly functional binding sites.After selection of antibodies with pairing-compatible variable regionsas described above, the antibody variable region genes can be clonedinto expression vectors that will direct the expression of an antibodyof the desired format, e.g., IgG, IgA, IgM. In one embodiment, describedis the production of mixtures of antibodies through the co-expression ofvariable region genes operably linked to constant region genes, in whichthese variable region genes encode different antibodies withpairing-compatible variable regions. Without the selection ofappropriately pairing antibodies with pairing-compatible variableregion, co-expression would lead to the formation of a mixture ofantibodies with many non-functional heavy-light chain combinations. Whenappropriate pairing-compatible variable regions have been defined, ahigh level of functional antibody combining sites will arise. In oneembodiment, the heavy chain variable region is operably linked to thefirst domain of the heavy chain constant region, followed by a hingeregion, followed by the remaining domains of the heavy chain constantregion. The variable region of the light chain on the other hand isoperably linked to an appropriate constant domain of the kappa or lambdafamily.

In certain embodiments, the pairing-compatible variable region is anidentical light chain. In that case the co-expression of this lightchain and, for example, two different heavy chains derived fromantibodies with as pairing-compatible variable region the full lightchain, in the same cell will yield a mixture of the two expectedbivalent molecules and one bispecific molecule. Similarly, whenco-expressing this light chain with more than two heavy chains derivedfrom antibodies that all have functional antigen binding sites whenpaired to that same light chain, the mixture will contain in a certainfraction each of the bivalent molecules, and a number of bispecificmolecules with combinations of all binding sites, e.g., three when threeantibody heavy chains are introduced, six when four antibody heavychains are introduced, ten when five antibody heavy chains areintroduced, etc. In this case, the affinity of the monomeric bindingsites in these various species is expected to be very similar to theaffinity of the original binding sites. In another embodiment,antibodies share a pairing-compatible variable region, but the sequenceof this element is different between the two antibodies and, uponswapping, the affinity of one or both of the antibodies may be altered.If such antibodies are used for co-expression, the final antibodymixture will contain antibodies with the original and the alteredbinding affinity in all of the species that were mentioned above. Insome embodiments, such antibodies share a compatible common light chainIn another embodiment, antibodies share a compatible common heavy chainThe expression levels of the individual components can be chosen or canbe manipulated to alter the fraction of the species of antibodiescontaining that component.

2. Protein Mixtures with Optimally Paired Variable Regions

Using the methods described herein, antibodies with a pairing behaviorsuitable for the preparation of well-defined biopharmaceutical mixturesare obtained. Traditionally before use for human therapy, protein drugsare expressed and purified to homogeneity, consisting of one majormolecular species. In some cases, therapy is more efficacious withcombinations of proteins or other drugs. Embodiments include methods tomake a proteinaceous mixture that will contain at least two majormolecular species, composed of at least three variable regions, and suchthat some variable regions pair to form a functional binding site. Thelarge-scale manufacturing of the proteinaceous mixture is a prerequisitefor their clinical use, and a simple purification procedure is animportant feature of the development process. The presence ofinappropriately paired variable regions would inevitably lead to a morecomplicated purification procedure. In one embodiment, the genesencoding the components of the two proteinaceous compounds areco-expressed in the same host cell, and the different major molecularspecies that are present in the mixture and have a functional bindingspecificity purified using biochemical/biophysical techniques well knownin the art. In one embodiment, the method is used to make a mixture of adefined number of antibodies. The major molecular species that compriseone or more different binding specificities could share a minimalproportion of their encoding genetic information (e.g., an Fc region, acommon tag, or another shared domain or feature); such shared featurewill provide a common mechanism/assay for following the individualcompounds in the mixture. In another embodiment, the major molecularspecies are co-purified due to a similar biophysical/biochemicalbehavior, or due to a shared domain that mediates co-purification (e.g.,an Fc). In another approach, the major molecular species are fused to asubunit of a protein such that they can multimerize with each other(e.g., CH2-0H3 region). Also provided are biopharmaceutical mixturesproduced using this method. In some embodiments, the application is theco-expression of antibodies, with the choice of the V-genes and pairingbehavior between VH and VL domains such that mainly or only functionalbinding sites are made, and the purification of the mix can occur viathe shared feature, an Fc region. Methods for purification ofimmunoglobulin are well known in the art, including protein A, protein Gand other affinity matrices. Other proteinaceous mixtures that could beenvisaged to have paired variable regions are fusion proteins betweenantibodies or antibody fragments and other molecules, single domainantibodies derived from camel, llama or engineered single domainantibodies from murine or human variable region genes, receptorextracellular domains, peptides, proteins equipped with an engineeredbinding site, or cytokines. In some embodiments, the proteinaceouscompounds share a feature (like by further fusion to an immunoglobulinFc region; methods well known in the art), such that they can beco-purified using the same procedures. The optimal pairing of thevariable regions in the different proteinaceous compounds will also leadto an optimal level of functional binding sites on these compounds, thusminimizing the number of purification steps required to obtain theactive component of the protein mixture.

3. Selecting Antigen-Specific Proteinaceous Compounds using Mixtures ofEncoding DNA

In certain embodiments, the proteinaceous compounds are antibodies.Antibodies are identified in collections or pools of genetically diverseantibodies, in which the pairing of the variable genes is optimized insuch manner that upon co-expression of at least two antibodies insidethe same cell an optimal pairing arises, providing a maximal amount offunctional binding sites. In some embodiments, the pairing of allbinding sites is optimized due to the use of a shared variable regiongene, for example the light chain. The diversity of the other elementsin the library will be such that antibodies with high affinity can stillbe selected. Due to this choice of the genetic make up of the variableregions, the pairing of the antibody variable regions will be such thata very high level of functional binding sites will be present whenmultiple variable regions forming more then one antibody binding siteare contacted with one another, for example, when expressed in the samecell. In one embodiment, first a library or collection of differentantibody heavy chain genes is made, and cloned into an eukaryotic cellexpression vector. This library is introduced into host cells in such amanner that each host cell will be making multiple different antibodyheavy chains. In particular embodiments, “anti-repressor elements”(Kwaks et al., 2003, Nat. Biotechnol. 21:553) are cloned at one or bothends of the antibody heavy chain gene. Such elements confer stable andhigh level expression of a given transgene as shown in this citation,and herein describe is its use to mediate stable and high levelexpression for each individual copy of the transgene (see, also below).

In one embodiment, depicted in FIGS. 7 and 8, the variable region orregions with optimized pairing behavior for the other variable regionsis or are also genetically encoded in an appropriate expression vector,and introduced into the host cell, either before, during or after theintroduction of the other variable region. The expression cassette withthe variable regions can also be part of a viral system such that highlevels of transfection/infection efficiency can be achieved. In the casethat the pool of first variable regions are antibody heavy chains, thesecond variable region with optimized pairing behavior can be one ormore light chains The host cells which are transfected with bothpartners of the pairing, e.g., the mix of antibody heavy chains and setof light chains, are expanded and grown under conditions which allow theexpression of heavy chains and light chains In some embodiments, onlyone light chain is used, as exemplified in FIG. 7. For example, theexpansion can occur in tissue culture wells, in such a manner that thetissue culture wells will contain between 10-1000 different originallytransfected clones, each of the clones expressing multiple pairings ofthe antibody variable regions. Antigen-specific antibodies can beretrieved amongst these clones and wells by various methods, for exampleby ELISA or equivalent test of the antibody mixtures of each well (see,also earlier description of binding assays). If stable transfection isused, with the possibility to select transfected cell lines for stablyintegrated copies of the antibody encoding DNAs, the relevant antibodyor antibodies may be cloned via limiting dilution. Alternatively, theDNA encoding the relevant antibody variable genes can be retrieved byamplifying and sequencing the antibody genes from the cells in the wellusing methods know in the art. If required, the antibody-heavy chainencoding DNA can be also amplified, recloned for expression in the samesystem, the DNA amplified and then used to repeat the transfection,expression and screening experiment. With this cycle of transfection andscreening, after a few rounds, an antigen-reactive antibodies startdominating the population. At every round, the complexity of the mixtureproduced by an individual cell can be reduced by reducing the complexityof the DNA introduced into the cell, to eventually become a oligoclonalpopulation. From the transfected wells, the antibody's V-gene can berescued directly (e.g., via PCR) and further analysis and/or screeningin this system, eventually at conditions that provide expression of themonoclonal antibody. Alternatively, the variable regions from reactivewells can be cloned into other systems for rapid screening of thebinding specificity of the individual pairs of variable regions, e.g.,via bacterial expression of antibody fragments or whole IgG, expressionin other hosts, via in vitro display methods, bacteriophage displaymethods etc.

In certain embodiments, the heavy chains may be secreted by the hostcell into the supernatant, where they can be reconstituted intofunctional antigen binding fragments, by the addition of and pairingwith a partner light chain This can be a small family of related chains,but may be one chain only. In this approach, cells are used that do notprevent secretion of the non-paired heavy chain. This embodiment isdepicted in FIG. 8. Drosophila S2 cells have been described that containa BiP (Binding Protein) homologue, hsc72, that specifically interactswith immunoglobulin heavy chains, but does not prevent their secretion.Alternatively, the heavy chains will need to carry amino acid mutationsin such a manner that cells that normally retain heavy chains when theyare not paired to light chains, will not mediate retention anymore. Forexample, mutations can be provided for or, selected within, the majorrecognition sites for BiP sites which are located in the heavy chain CH1domain. For example, the CH1 domain can be replaced (e.g., by a CL orCH3 region) as long as the light chain can pair with this form of themolecule (or other variants, see also section on antibody cross-overvariants), or mutated to avoid retention by BiP. The results of suchvariations are that the different heavy chains are secreted by the hostcell. The chains are then reconstituted with one or more partner chainscarrying the partner variable region(s). Methods to establish this havebeen extensively reviewed in literature on the biochemical analysis andassembly of antibody molecules. Antigen-reactive variable region pairscan be identified in the same way as described for the other embodiment.

In yet another embodiment, the first partner of the two paired variableregions (such as the heavy chain for an antibody) is anchored onto aeukaryotic cell surface, and the other variable region provided byexpression in the same host cell or via reconstitution on thecell-surface. This set-up allows a direct screening for antigen-bindingon the host cell surface, for example, via flow cytometry withfluorescently-labeled antigen, or a direct selection, for example, viacell sorting methods.

Methods to identify antigen-reactive antibodies from B-cell populationshave been described in the literature and can be applied to thesetransfection-based systems also. In such described systems, randomcombinatorial diversity is sampled, and antibody variable gene pairingis also not optimized or biased. Use of such random combinatorial pairsof variable regions does not guarantee that upon production of anantibody mixture, the pairing will be optimal; on the contrary,mispaired variable regions will be a substantial fraction of theproduced proteinaceous compounds. This random combinatorial diversity islimited by reducing the diversity of one of the variable region genes.The diversity that is present in the resulting paired repertoireoriginates mainly from one of the variable regions. For example, it maybe one or a small set of light chains. As a consequence, in theiterative process of selecting the antigen-reactive variable regions,only one of the two partners of the pair will need to be identified. Itis not necessary to retrieve both the heavy and light chain variableregion sequence from the same cell. Another important difference is thatmultiple antibody genes are introduced and expressed from the same hostcell. When using random diversity, such a feature would lead to amultiplication of the diversity and reduction of the quantity of theindividual combinations to the extent that detection let alone cloningof the responsible antibody gene combination would become verydifficult, if not impossible. Consider the case in which the cell wouldbe making multiple combinations of heavy and light chain pairs, then thechance to retrieve the correct combination of the antibody that mediatesantigen reactivity, would be become smaller as the cell is making ahigher number of different chains. If the cell were expressing tendifferent heavy and light chains, the combinatorial diversity generatedby this one cell would be a 100 different types of antibody bindingsites; only 1/10 of the antibody variable genes amplified from such cellwill be the relevant one, thus the chance to be able to clone thecorrect antibody genes is very low. As a consequence of this reducedcombinatorial diversity, there will also be a higher quantity of each ofthe individual antibodies, which makes a more sensitive detectionpossible. Thus, the expression of the different antibodies in the samehost cell is a desired feature. First as explained above, it is animportant feature for the antigen-selection system to findantigen-reactive antibodies when using transfected cell populations.Second, the methods are directed towards the production of mixtures ofproteins and more in particular, antibodies or their fragments, whichrequires optimal pairing of the variable regions, in particular, whenproducing such mixtures by co-expression in the same host cell. In themethod described above, co-transfection of variable region genes insidethe same cells leads to the expression of multiple antibodies in thesame host cell. The methods are thus useful to select individualantibody variable region pairs that are reactive with a given targetepitope, but also to select a mixture of different variable region pairsall reactive with a given target epitope (in the process of thescreening, multiple antibody variable region pairs will be selected oridentified, but when iterating the process, these antibodies are likelyto be eventually mixed and end up in the same host cell). Further if thescreening or selection of the mixture is carried out with targets withmultiple epitopes, or multiple targets, the mixture can also containantibodies to multiple epitopes or targets, yet withco-expression-compatible pairing of the variable region genes.

The methods are also suitable for the screening of mixtures of proteinswith paired variable regions that have a defined binding specificity(FIG. 9). The genes encoding these compounds are introduced as a mixtureinto a host cell as above (in FIG. 9 examples is given of ten differentantibodies), and individual clones that have integrated some or multiplecopies of the genes encoding the various variable regions expanded. Inthe way described above, applied to antibodies, the supernatants of theresulting cell lines are screened for reactivity towards the variousantigens. The levels of each of the individual antibody pairs may vary,and, when the antibody format is the IgG isotype, also the level of thebispecific antibodies resulting from the co-expression may be highlyvariable. Cells that secrete the mixture comprising the desiredcomposition are identified and used as a stable production host for thismixture. Provided is a method to quickly screen hundreds of mixtures ofdifferent antibodies. The optimized pairing of the heavy and lightvariable regions will secure a high level of functional binding sites inthe antibodies present in such mixtures.

4. Antibody-Based Compounds with Paired Variable Regions and Cross-Overor Mutations in the Constant Regions

The pairing of the variable and constant regions of an antibody can befurther engineered by crossing-over domains. Antibodies are made by“crossing-over” or swapping or replacing elements within the Fab regionof the antibody (or the antibody heavy chain Fd region and the antibodylight chain region), and combining the appropriate elements to establisha binding site in the context of an immunoglobulin molecule (examplesare given in FIG. 10). In its simplest format, the L chain and H chainFd region are swapped. A VL-CL-hinge-CH2-CH3 chain is thus paired to aVH-CH1 domain. In a second format, the constant region genes between Hand L are swapped. In another form, the CH1 is replaced by a CL. Inanother form, the VH and VLs are swapped. In another form, one or moreof the CDR regions between VH and VL are swapped. The pairing efficiencycan be monitored in such cross-over variants, such that suitablecombinations of non-cross-over antibodies with cross-over antibodies, orcombinations of different cross-over antibodies, can be used to mediateoptimal pairing when making mixtures of at least two antibody molecules(with antibody also including here cross-over variants as describedabove). In another form, the effect of mispairing between different VHsand/or VLs is reduced by linking the VH and VL via a linker to asingle-chain Fv variant, which will favor the association between thesetwo domains. Alternatively, the pairing between variable regions can bemanipulated by the introduction at the appropriate positions ofcysteines which upon pairing of the variable heavy and light variabledomains can form a disulphide bridge. Also provided are methods forselecting antibody fragments that will bind antigen in an appropriatecross-over format, by selecting from appropriately formatted libraries,or by screening one or more antigen-binding antibodies for the activityin the cross-over format. Antibodies in which the CH1 domain is not partof the heavy chain may be secreted as free molecules not paired to lightchains, allowing alternative approaches for the production of antibodiesand new fusion formats. Antibodies in which the variable regions areswapped may be functionally non-equivalent and yield a more diverse,unnatural or different spectrum of antigen-binding or biologicalactivity (the positioning of the heavy and light chain variable regionsis expected to not always be completely equivalent). Besides effects ofthe exchange of the heavy and light chain genes on affinity and/orspecificity, the swapping may alter the antibody flexibility and impactthe biological behavior. Finally, an antibody binding site with chimericVH-VL regions (with CDR or FR regions swapped between the two variabledomains) may also yield an alternative, possibly larger but structurallynon-overlapping set of antibody paratopes.

Second, selective engineering of the constant regions or the interactionof variable regions with constant regions may also affect the pairingbehavior of the variable region genes. By modifying the antibody heavychain constant region, the fraction of functional bispecific antibodiescan be increased or decreased. In this approach, antibody heavy chainscan be engineered to drive hetero- or homodimerization. This can be doneby introducing sterically complementary mutations in the CH3 domaininterface, for example, as has been described in the literature forincreasing the percentage of functional bispecific antibodies in themixture of antibodies arising from the co-expression of two heavy andtwo light chains The pairing of the antibody binding site variableregion may thus be influenced by the pairing of variegated constantregions, of heavy and light constant region domains.

5. Extracellular Pairing of Proteinaceous Mixtures

Provided are methods for making whole antibodies using an in vitropairing procedure of heavy and light chains produced in different hostcells. In one embodiment, one of the two antibody chains is expressed ina first host cell and the other chain is expressed in a second host cell(FIG. 11, section A). The antibody chains are then brought togetherunder conditions in which pairing of the two domains will occur, thusoutside of the cell. In one embodiment, the pairing occurs in vitro,with purified chains and under conditions that are optimized for thepairing of the desired variable regions. In another embodiment, theexpression occurs via the use of one or two dummy-chains, temporarilypaired to the respective variable regions, removing the dummies fromtheir partner via a mild and controllable process, and pairing theappropriate unpaired variable regions to one another to form afunctional binding site. In one embodiment applied to antibodies, thisassociation is made easier by using heavy-light chain pairs mutated inone or the other chain to facilitate the process of the pairing, e.g.,mutated in the cysteine residue that normally forms the bridge between Hand L chains (either both mutated, for example, to Ser, or only onemutated and not the other), or mutations that have altered the affinityof one chain for the other or mutations in the dummy chain used for thetemporary pairing, in particular, the one that pairs with the heavychain; thus such dummy light chain will pair with a native, non-mutatedheavy chain, and may carry mutations such that it can be readily removedfrom the purified antibody.

An extension of this concept is that it is possible to produceantibodies using universal antibody chains (FIG. 11, section B).Provided are methods for expressing a shared, invariant variable regioncontained into the appropriate chain format (e.g., a VL-CL light chain)in a given host cell, and the other chain (e.g., a heavy chainconsisting of VH-CH1 or VH-CH1-hinge-CH2-CH3) that is dominant in orprovides most or all of the specificity, in another host cell. Forproduction of two antibodies, three chains need to be made, which can beassembled in vitro to form two different antibodies. For example, if thelight chain is identical, only one VL-CL domain will have to be made,and two VH-containing heavy chains. These can then be assembledextracellularly, for example, in vitro. Pairing of the variable regionswill have to be optimal such that the proteinaceous mixture yields ahigh level of functional binding sites. The light chain can be useduniversally for all antibodies that will accommodate it (and antibodiesaccordingly selected if required). The heavy chain can be expressed inmammalian cells to provide a suitable glycosylation; for the lightchains any suitable expression host cell can be chosen. When using thecross-over variants described in the previous section, in which thelight chain is fused to the hinge and Fc, and the heavy chain variableregion is provided as the lightest chain (as VH-CH1 or VH-CL), animportant advantage of this set-up is apparent: the light chain fused tothe Fc (depicted as “constant” chain in FIG. 11, section B), with itsfunctionally important glycosylation features, can be made as theuniversal chain. The heavy chain can carry the dominant features for thespecificity, and a mixture of heavy chains which will mediate differentbinding specificities can now be made in a different host cell that doesnot need to provide glycosylation. Such a feature makes the productionof mixtures possible in two steps: a cheaper prokaryotic expression canbe used to make mixtures of variable regions each encoding a uniquebinding specificity, while the more expensive production of the othervariable region that also requires most fine analysis, can be done in aeukaryotic host. All antibodies that can pair with the latter variablegene without inflicting their overall specificity and affinity, can beproduced by extracellular pairing with the same universal chain. Thelatter can be designed to be optimized for pharmaceutical applications:a broadly expressed, relatively common variable region, with a minimalnumber of MHC Class II epitopes, of human origin, and germ line insequence. This procedure of mixing can be done with separate heavy chainmixtures or with a mix of the different heavy chains; when applied tothe IgG format as depicted in FIG. 11, section B, the result is anantibody mixture without or with bispecifics, respectively. Manualmixing and pairing of variable region genes further provides much morecontrol over the pairing, it can be done in a stepwise manner, perantibody, per group of antibodies etc. For some applications, forexample, where there is an absolute necessity to avoid the formation ofbispecific antibodies in a complex mixture with three or moreantibodies, this method has an advantage over the cell line-basedapproach.

6. Controlling the Expression of Variable Regions in the Context of theProduction of Multiple Pairing Variable Regions in the Same Host Cell

Nucleic acid molecules encoding variable region, e.g., from antibodies,can be co-expressed in the same cell to make mixtures of differentfunctional binding sites. With appropriate pairing behavior, a highlevel of functional binding sites will be present. It will however alsobe important to control the expression of the individual variableregions and their expression ratios, because this will effect thecomposition of the final antibody mixture. The expression level and thestability of the expression is a function of the site of integration ofthe transgene: if the transgene is integrated close to or withininaccessible chromatin, it is likely that its expression will besilenced. Described is the use for the production of mixtures ofantibodies in the same cell, of elements that, when flanking theantibody genes, will increase the predictability of the expressionlevel, the yield, and improve stability. Such elements can, for example,do this by counteracting chromatin-associated gene repression. Suchanti-repressor elements provide a high level of predictability ofexpression, high levels of expression and stable expression overtime, ofthe antibody mixture (Kwaks et al., 2003, Nat. Biotechnol. 21:553). Suchelements confer stable and high level expression of a given transgene asshown in this citation, and herein described is its use to mediatestable and high level expression for each individual copy of a mixtureof transgenes, encoding multiple variable regions. A variety of suchelements and other systems to achieve a similar result have beenidentified in the art, including Locus control regions (LCRs), chromatinopening elements, artificial chromosomes (e.g., ACE technology fromChromos Molecular Systems Ltd.), and Ubiquitous Chromatin OpeningElements. For example, LCRs are transcriptional regulatory elementswhich possess a dominant chromatin remodeling and transcriptionalactivating capability conferring full physiological levels of expressionon a gene linked in cis, when integrated into the host cell genome. Inthe following section, “anti-repressor elements” are described butother, different control elements such as the ones mentioned andinasmuch as they provide the opportunity to regulate the high-levelexpression of multiple genes, may be equally suitable to achieve acontrolled expression of the different variable regions.

In one embodiment, antibody mixtures are made from variable region pairsin which one dominates the binding, and the other is a shared variableregion. In certain embodiments, the first variable region one is theheavy chain, and the second is the light chain. In certain embodiments,at least one of the antibody heavy chains is flanked by oneanti-repressor element, or by two identical or two differentanti-repressor elements located at either end of the heavy chain gene;in another embodiment, more than one or possibly all of the heavy chaingenes that need to be expressed are flanked by anti-repressor elements.In one embodiment, the heavy chains are based on the same plasmid, inanother they are on separate plasmids. In another embodiment, CHO cellsare used as host; in another embodiment, PER.C6® cells are used.

The manufacture of mixtures of antibodies expressed in the same cellline will require appropriate variable region pairing and also a stableexpression level of all of the antibody chains involved, as well as astable ratio of the various chains, in such manner that the resultingantibody mixture after manufacture even at GMP conditions, has a stablecomposition. Such stable compositions can then translate into stablebiological activity and stable toxicity profile. If the expression ofonly one antibody chain would change, it could affect the compositionand, therefore, also alter its biological activity. The provision ofelements that yield a more predictable and copy-number associatedexpression level is also important to build cell lines that expresssimilar or even equimolar levels of different antibodies. If, forexample, five antibody heavy chains have to be expressed, it will bevery difficult to build a cell line that expresses all of these chainsat similar quantities when using a random integration and selectionapproach without the anti-repressor elements. By using such elements, ahigher copy number of antibody chains can be introduced withoutcompromising the stability of the resulting cell line. Thus, multipleantibody heavy chains can be introduced, where the number of integratedcopies for each heavy chain will also to some level reflect its absoluteexpression level. With such elements it will be much easier and morerapid to alter the ratios of expression levels between the heavy chains,for example, by manipulating the ratios of the DNAs encoding the heavychains at the time of the transfection.

This also explains embodiments including incorporation of suchanti-repressor elements in vectors to be used for creating antibodylibraries and select antigen reactive antibodies from these pools (see,section 4); anti-repressor elements which may be inserted in theexpression vectors that incorporate the heavy chain, on FIGS. 7, 8 and9.

7. Expression Systems for Multiple Variable Regions in the Context ofthe Production of Multiple Regions in the Same Host Cell

When expressing multiple variable regions inside the same cell, maximalproductivity will be achieved only if the partners that need to bepaired are co-expressed at an equivalent level, such that there islittle chance on what is essentially waste: the non-paired variableregion. The composition of the mixture is influenced by manipulating anyone of the parameters that affect the expression level achieved in thehost cell. The expression level of a given component is a function ofmany factors including the regulatory sequences that drive theexpression of the component, when the component is a heavy chain alsothe expression levels of the light chains, the choice of the host cell,the method of expression (transient or stable), and, for stableexpression, the copy number and site of integration. The expressionlevels can further be affected by many parameters including choice ofthe transcriptional regulatory elements (including choice of promoter,enhancer, insulators, anti-repressors, etc.). The expression of the twolight and heavy chains of the antibodies that are to be assembled fromthe mixture of the chains can be done independently for each of thechains, or made dependent from each other.

The expression vector or vectors comprising the antibody genes ofinterest contain regulatory sequences, including, for example, apromoter, operably linked to the nucleic acid(s) of interest. Largenumbers of suitable vectors and promoters are known to those of skill inthe art and are commercially available for generating the recombinantconstructs herein. Appropriate cloning and expression vectors for usewith prokaryotic and eukaryotic hosts are described by Sambrook et al.,in Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporatedherein by reference. The following vectors are provided by way ofexample. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, and pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI,pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). Promoterregions can be selected from any desired gene using CAT (chloramphenicoltransferase) vectors or other vectors with selectable markers. Twoappropriate vectors are pKK232-8 and pCM7. Particular bacterialpromoters include lacI, lacZ, T3, T7, gpt, lambda P, and trc. Eukaryoticpromoters include CMV immediate early, HSV thymidine kinase,Elongation-factor-la, early and late SV40, LTRs from retrovirus, mousemetallothionein-I, and various art-known tissue-specific promoters.Methods well known to those skilled in the art can be used to constructvectors containing a polynucleotide as described herein and appropriatetranscriptional/translational control signals.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and also any necessary ribosome-binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences.Expression regulatory sequences may comprise promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of the sequences. Alternatively, sequences which affect thestructure or stability of the RNA or protein produced may be replaced,removed, added, or otherwise modified by targeting, includingpolyadenylation signals, mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules. In addition to the nucleicacid sequence encoding the diversified immunoglobulin domain, therecombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017). For example, typically the selectable marker gene confersresistance to drugs, such as G418, hygromycin or methotrexate, on a hostcell into which the vector has been introduced. Examples of selectablemarker genes include the dihydrofolate reductase (DHFR) gene (for use indhfr⁻ host cells with methotrexate selection/amplification) and the neogene (for G418 selection).

In an exemplary system for recombinant expression of a modifiedantibody, or antigen-binding portion thereof, a recombinant expressionvectors encoding at least one antibody heavy or light chain isintroduced into dhfr⁻ CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy or light chain gene is operatively linked to enhancer/promoterregulatory elements (e.g., derived from SV40, CMV, adenovirus and thelike, such as a CMV enhancer/AdMLP promoter regulatory element or anSV40 enhancer/AdMLP promoter regulatory element) to drive high levels oftranscription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy or light chains.In many instances the expression vector may contain both heavy and lightchain genes, and co-transfection will lead to the production of intactantibody, recovered from the culture medium. Standard molecular biologytechniques are used to prepare the recombinant expression vector,transfect the host cells, select for transformants, culture the hostcells and recover the antibody from the culture medium. For example,some antibodies can be isolated by affinity chromatography with aProtein A or Protein G.

The host may also be a yeast or other fungi. In yeast, a number ofvectors containing constitutive or inducible promoters may be used. Fora review, see, Current Protocols in Molecular Biology, Vol. 2, Ed.Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13(1988); Grant et al., Expression and Secretion Vectors for Yeast, inMethods in Enzymology, Ed. Wu & Grossman, Acad. Press, N.Y. 153:516-544(1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3(1986); Bitter, Heterologous Gene Expression in Yeast, in Methods inEnzymology, Eds. Berger & Kimmel, Acad. Press, N.Y. 152:673-684 (1987);and The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern etal., Cold Spring Harbor Press, Vols. I and II (1982). The host may alsobe a prokaryotic organism, such as E. coli. As a representative butnonlimiting example, useful expression vectors for bacteria can comprisea selectable marker and bacterial origin of replication derived fromcommercially available plasmids comprising genetic elements of the wellknown cloning vector pBR322 (ATCC 37017). Such commercial vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega, Madison, Wis., USA).

Introduction of the recombinant construct into the host cell can beeffected, for example, by calcium phosphate transfection, DEAE, dextranmediated transfection, or electroporation (L. Davis, et al., BasicMethods in Molecular Biology (1986)).

DNA encoding the antibodies is readily isolated and sequenced usingconventional procedures for cloning, DNA preparation and sequencing asdescribed by Sambrook, et al., in Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosureof which is hereby incorporated by reference. For sequencing,oligonucleotide probes can be used that are capable of bindingspecifically to genes encoding the heavy and light chains of antibodiesor to the vector sequences surrounding the gene fragments, and the DNAsequence determined by dideoxy-based sequencing (F. Sanger, et al.(1977) PNAS 74:5463-5467). Once isolated, the DNA encoding appropriateregions of the antibody may be placed into expression vectors, which arethen transfected into host cells. The host cell can be a highereukaryotic host cell, such as a mammalian cell, a lower eukaryotic hostcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

In some embodiments, antibodies with pairing-compatible variable regionsare produced in mammalian cells. Examples of mammalian host cells forexpressing the clone antibodies or antigen-binding fragments thereofinclude Chinese Hamster Ovary (CHO cells) (including dhfr⁻ CHO cells,described in G. Urlaub et al. (1980) PNAS 77:4216-4220), used with aDHFR selectable marker, e.g., as described in (R. J. Kaufman et al.(1982) J. Mol. Biol. 159:601-621), lymphocytic cell lines, e.g., NSOmyeloma cells and SP2 cells, C127, 3T3, CHO, human epidermal A431 cells,Jurkat, U937, HL-60, mouse L-cells, Baby Hamster Kidney cells, COS orCV-1 cells, PER.C6® cells (M. G. Pau et al. (2001) Vaccine19:2716-2721), other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, and a cell from a transgenic animal, e g , atransgenic mammal. For example, the cell is a mammary epithelial cell.Other cell types suitable for expression, in particular, for transientexpression, are simian COS cells (Y. Gluzman (1981) Cell 23:175-182),and Human embryonic Kidney cells of lineages 293, 295T and 911 (Hek293,295T, 911).

Alternatively, it may be possible to produce the antibody as fragment oras whole antibody in lower eukaryotes such as yeast or in prokaryotessuch as bacteria (L. C. Simmons et al. (2002) J. Immunol. Methods263:133-147). Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the full antibody ismade in yeast or bacteria as IgG, it may be necessary to modify theprotein produced therein, for example, by phosphorylation orglycosylation of the appropriate sites, in order to obtain thefunctional protein. Such covalent attachments may be accomplished usingknown chemical or enzymatic methods. Recombinant polypeptides andproteins produced in bacterial culture are usually isolated by initialextraction from cell pellets, followed by one or more salting-out,aqueous ion exchange or size exclusion chromatography steps. In someembodiments, the template nucleic acid also encodes a polypeptide tag,e.g., penta- or hexa-histidine. The recombinant polypeptides encoded bya library of diversity strands can then be purified using affinitychromatography. Microbial cells employed in expression of proteins canbe disrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

Described herein is a method to directly relate the expression of thetwo partner variable regions that are required to pair in such mannerthat there is minimal waste (FIG. 12). The nucleic acid moleculeencoding the first variable region is cloned into an expressioncassette, such that it will be under the control of a given promoter(typically the strong CMV promoter or other), and such that its codingsequence is followed by an Internal Ribosome Entry Site (IRES) and thecoding sequence of the transactivator of the tet responsive element(TRE) fused to the activation domain of the herpes simplex VP16 protein(tTa). The nucleic acid molecule encoding the second variable region iscloned into an expression cassette such that its expression is regulatedvia an inducible promoter, for example, the tet responsive element(TRE), existing of seven copies of the prokaryotic tetracycline operatorsite fused to a minimal CMV promoter. When introducing both expressioncassettes into the same cell (on different vectors or on same vectors,at the same time or one before the other), the following relationbetween the expression of the two variable regions will exist:expression of the first variable region, which is under control of, forexample, a constitutive promoter, will lead to the expression of the tTaprotein. This protein activates the TRE-based promoter which will drivethe expression of the second variable region. Thus, the production ofthe second variable region is now dependent on the production of thefirst variable region. If these regions are required to pair, theproduction of the individual components of the pairing can be madedependent.

When antibodies of the IgG-type are produced via a heavy and lightchain, the production of the light chain can be made dependent on theproduction of the heavy chain Consider the embodiment including theproduction in the same host cell of a mixture of antibodies which allshare a pairing-compatible light chain. The light chain gene is clonedunder control of the TRE element, while the heavy chains are allprovided with the IRES and tTa gene, as described above. In the hostcell, every individual heavy chain that is expressed will then triggerthe production of more partner light chain. This is important, becausewith multiple heavy chains being expressed, it is likely that the levelof light chain may become limiting, and that the excess of unpairedheavy chain will induce possible toxicity in the host cell (as has beendescribed for B-cells). This concept is also applicable to theembodiment described in section 4, for the selection of antigen-reactiveantibodies from pools made in eukaryotic cells. Otherpromoter-transactivator systems have been described and are applicablein this concept also. In the same application field, in those caseswhere the ratios of two particular heavy chains need to be controlled orfixed, this method of dependent-expression may be used to link theexpression of two heavy chains

Generally, a large number of suitable vectors and promoters are known tothose of skill in the art and are commercially available for generatingthe recombinant constructs described herein. The following vectors areprovided, by way of example, for the expression in eukaryotic cells oftwo or three antibodies that share a light chain sequence. The antibodychain encoding genes are cloned into expression cassettes that provideall regulatory and secretion signals which are typically used forantibody expression, as depicted in FIG. 20. In a first embodiment, theexpression of multiple antibody heavy chains is made dependent on oneanother in the following way. In the first embodiment, the nucleic acidencoding the first heavy chain (H1) is cloned into an expressioncassette, such that it will be under the control of a given promoter(typically the strong CMV promoter or other), and such that its codingsequence is followed by an Internal Ribosome Entry Site (IRES). This isimmediately followed by a second antibody heavy chain coding region (H2,as depicted in FIG. 21). The P1 promoter will now drive the expressionof H1 and H2, leading to an approximate 1:1 expression ratio betweenthese two proteins; often though the second coding region is slightlyless well expressed. Thus, if the expression ratio has to be steeredtowards a predefined range, the use of IRES sequences is particularlyuseful. This predefined range is influenced among other factors by thenature of the IRES sequence, and different IRES sequences will mediatedifferent final ratios. Similarly, the expression ratio between threeantibody heavy chains can be linked to one another by using atricistronic expression cassette, in which the previous describedcassette is followed by another IRES and Heavy chain coding region.Examples of tricistronic expression systems and of IRES sequences andconfigurations are described for other systems in the literature (Li etal., J. Virol. Methods 115:137.44; When et al., Cancer Gene Therapy8:361-70; Burger et al. 1999, Appl. Microbiol. Biotechnol. 52:345-53).In these embodiments, the shared antibody light chain can be provided ona separate expression plasmid, on one or more of the vectors that carryon or multiple the antibody heavy chains, or can be already expressed bythe host cell used for the transfection with the heavy chain expressionvector or vectors.

In another embodiment, antibody heavy genes are sequentially transfectedinto the host cell. First, the embodiment for libraries of cells thatproduce mixes of two antibodies are considered. Cells are transfectedwith the two antibody genes cloned into different vectors but thetransfection is done sequentially in time. For example, the antibodyheavy and light chain encoding regions of the first antibody areintroduced into the host cell, and stable transfectants expressing thisantibody identified and isolated. The antibody genes encoding a secondantibody, in which the variable regions are pairing-compatible, aretransfected into the host cell that already expresses the first antibodygenes at high level. This procedure of carrying out sequentialtransfections (and if appropriate selections of integration in between)is also suitable for making collections of mixture with up to four tofive different antibodies. To increase the number of cell clonesexpressing multiple antibodies, the vectors carrying the genes encodingthe antibody genes, also carries a unique selection marker, such thattransfected cells that have integrated the vector sequence can bereadily selected and antibody expressing clones identified. As analternative embodiment for making cells that express multiple antibodieswith compatible pairing, the following procedure is used. First, asbefore, cell clone is produced that expresses one set of antibody chains(this can be one H and one L or multiple H and one L, for example) andis selected on the basis of a first selection marker. In parallel, acell clone is produced that expresses another subset of antibody chains(for example, one or more other H and one L) and that is selected on thebasis of a different selection marker (for example, neo, gpt, zeo, bdl,etc.). These cell clones are then fused and selected for the presence ofboth of the selective markers. Methods for cell fusion are extensivelydescribed in the literature and known to those working in the field;they are similar to those described in Norderhaug et al., 2002, Eur. J.Biochem. 269:3205-10. The hybrid cells have the potential to express allof the antibody chains. Similarly, this procedure can be repeated ifcollections of larger numbers of antibody chains have to be made.Further, the use of cell populations rather than cell clones, in thissequential transfection or cell-fusion approach, provides a method forachieving large collections of cells that express the antibody chains atdifferent ratios.

In one embodiment, the proteinaceous molecule's coding region or regionsare flanked by sequences that mediate site-directed integration into thehost cell genome (as depicted in FIG. 20). Without these, integration oftransgenes occurs at random and, usually, several copies of thetransgene are integrated at the same time, sometimes in the form of ahead-to-tail tandem, with the site of integration and the number ofcopies integrated varying from one transfected cell to another. The useof recombination sites as depicted in FIG. 20 allows the precise site ofintegration to be targeted by homologous recombination between vectorand host cell genome. This provides a means to insert the coding regioninto a site of high transcriptional activity, with the option to providea promoter in the transgene or use the one that is present at the siteof integration. With random or homologous recombination-mediatedinsertion of the antibody chain encoding nucleic acids is meant anyinsertion into the genome of the host cell, or into the nucleic acids ina subcellular organel, or into an artificial chromosome.

Some embodiments are to employ (per expression vector used in thelibrary construction) not more than three antibody heavy chain codingregions and may have two per vector. In certain embodiments, someplasmids do not contain more than three promoters and three IRESsequences and not more than six STAR or MAR elements. In some instances,the expression vector's size may be limited to 20 kb and if more thanfive binding sites are required in the mix, and these cannot befunctionally encoded in a plasmid that is less than 20 kb in size, touse two or more different plasmids.

MARs and STARs can be positioned on either side of the DNA sequence tobe transcribed. For example, the elements can be positioned about 200 bpto about 1 kb, 5′ from the promoter, and at least about 1 kb to 5 kbfrom the promoter, at the 3′ end of the gene of interest. In addition,more than one element can be positioned 5′ from the promoter or at the3′ end of the transgene. For example, two or more elements can bepositioned 5′ from the promoter. The element or elements at the 3′ endof the transgene can be positioned at the 3′ end of the gene ofinterest, or at the 5′ end of a 3′ regulatory sequence, e.g., a 3′untranslated region (UTR) or a 3′ flanking sequence. Chromatin openingelements can be flanking on both ends of the expression cassette (FIG.21, row D), or placed 5′ of the expression cassette (FIG. 21, row C). Inparticular, when multiple regulatory elements such as STAR and UCOs haveto be introduced into one and the same plasmids, elements may be usedthat have activity towards both ends of the element such that they canbe provided in the middle of an expression cassette (FIG. 21, row C).Since MARs have also been reported to function when co-transfected intrans with the transgene (Zahn-Zabel et al. (2001) J. Biotechnology87:29-42), they have the advantage that no DNA-cloning step is requiredto physically link them to SPCBP expression cassette(s). In that case,size of the MAR element or of the expression vector carrying the SPCBPcassettes is no longer a limitation. Nevertheless, MAR elements as smallas 1.3 kb have been described, thus multiple in cis inclusions arefeasible. MARs have also been reported to be added both in cis and intrans, and in this configuration increase expression levels ofantibodies in CHO cells 14-fold. One other function of theseelements—besides their effect on stability—is that they will alsoincrease the number of independently transformed cells that express theprotein and promotes higher amounts of the recombinant protein. Cloneisolation and production levels are overall higher, thus these elementsmay be used for making large collections of cell lines producingcompositions comprising multiple functional binding sites.

8. Proteinaceous Mixtures with Multiple Effector Regions and MultipleTypes of Binding Sites

The methods can be used to create compositions of proteinaceousmolecules that have multiple effector regions. In the case ofantibodies, compositions are included that display one or more antigenbinding regions in combination with two or more natural effectorregions. Examples are the effector regions encoded by IgG1 and IgG4,which have, for example, different binding regions for C1q and thevarious Fc-receptors based within their encoding constant regions. Suchmixtures may be clinically more effective than their mono-effectorcompounds: the mixture combines multiple and maximal natural effectors,which for various reasons are never present in the one natural antibodyisotype, and the mixture thus mimics much more closely the naturalpleiotropy of immune effectors that a single antigen/pathogen will evokewhen our immune system encounters it. Some formats are IgG1 and IgG4, orIgG and IgM, or IgG1 and Fab, or IgG and IgA, or IgA and IgM, orIgG1-cytokine fusion and alike. Instead of making such proteins indifferent hosts, the co-expression of such different antibody formats,all associated with the same binding site (or possibly multiple bindingsites but related to one target and, for example, to one disease orindication), allows the direct production of cocktails of antibodieswith different effectors. Such mixtures are more efficacious in theirbiological activity.

Besides antibodies, recent protein engineering techniques have allowedthe production of binding sites with predetermined specificity usingsimilar but also sometimes using very different structures. For example,antigen-specific ligands have been created using phage, bacterial,ribosomal or yeast display methods, from libraries of protein variants,in which the protein at some positions was variegated using random oroligonucleotide-based mutagenesis, but the main scaffold of the nativeprotein maintained in the variants. Proteins for which has been alreadyapplied include the protein Z domain of Protein A, a variety of Kunitzdomains, lipocalins, Green Fluorescent protein, one of the fibronectindomains, other domains of the immunoglobulin superfamily, and ankryns.Such antibody mimics are thus proteinaceous molecules with a non-naturalbinding activity, obtained, for example, by engineering into themolecule one or more residues or regions with variegated sequences, ateither defined or random positions, and identifying the molecule withappropriate antigen binding properties by screening or selectionprocesses. Examples of the processes are high-throughput screening forantigen binding by ELISA, or selection methods described in theliterature such as in vitro display methods such as ribosome andpuromycin display, cellular or viral display methods such as filamentousphage, lambda phage, bacterial, yeast, or eukaryotic cell display. Theresulting proteinaceous molecules with the new binding site is anantibody mimic in the sense that it will contain a binding region forantigen at the position where it was initially a variable region,similar to an antibody molecule with two variable regions.

9. Making Compositions of Multiple Proteinaceous Compounds withDifferent Binding Specificities.

Recombinant DNA technology provides methods well known in the art toclone the variable region genes, and produce cell lines expressing therecombinant form of the antibody. In particular, the properties ofantibodies are being exploited in order to design agents that bind tohuman target molecules, so-called “self-antigens,” and to antigens ofviral or bacterial diseases. For example, a number of monospecificantibodies have been approved as human therapeutics. These includeOrthoclone OKT3, which targets CD3 antigen; ReoPro, which targetsglycoprotein IIb/IIIa; Rituxan, which targets CD20; Zenapax andSimulect, which target interleukin-2 receptors; Herceptin, which targetsthe HER2-receptor; Remicade and Humira, which target tumor necrosisfactor; Synagis, which targets the F protein of respiratory syncytialvirus; Mylotarg, which targets CD33; and Campath, which targets CD52.

For many clinical applications the efficacy of the treatment wouldincrease if combinations of monoclonal antibodies are used. Anoligoclonal preparation can be made by mixing individual recombinantantibodies which each have been made by conventional procedures, whichincludes the expression and purification of the individual recombinantor hybridoma-derived monoclonal antibodies, and the subsequent mixing ofthese molecules. The pharmaceutical development of separately producedand then mixed monoclonal antibodies is inhibitively expensive.Recombinant monoclonal antibodies of the IgG isotype are commonly madeby co-expression of the nucleic acid sequences encoding the heavy andlight chain of the antibody in the same host cell, yielding a monoclonalantibody, bearing two identical binding sites. The production of severalantibodies from individual cell lines each making one antibody (and inwhich each cell line is controlled for stability of expression andconsistency), is not economical with present biotechnological productionmethods.

One approach to combining monoclonals is to combine the binding sites inone molecule, hence creating a multispecific antibody. This allows thetargeting of multiple epitopes on the same antigen, or of multipleantigens on the same target entity (e.g., a cell, a virus, a bacteria,an antigen), or of epitopes on different entities, providing a bridgebetween these entities. Of the multispecific antibodies, bispecificantibodies have been investigated the most, for targeting therapeutic ordiagnostic entities to tumor cells, e.g., a cytotoxic T-cell, an NKcell, a chelator that carries a radionuclide. But in the bispecificantibody the two binding sites are always covalently coupled to oneanother, which limits the flexibility and use of such compounds.Further, many of the recombinant bispecific antibodies (e.g., Fab-scFvfusions, diabodies, double-single-chain Fvs) lack the provision of theantibody's Fc region. Since Fc-dependent effector mechanisms such asADCC are important for the efficacy of many antibodies (e.g., Rituxanand Herceptin), it will be important to maintain this region in themultispecific molecule.

An alternative approach is to use polyclonal antibodies comprising theentire immune response of a host to an immunogen. Polyclonals derivedfrom the pooled serum from immunized animals or from selected humanshave been used therapeutically e.g., for passive or active immunization,e.g., anti-rhesus D, anti-digoxin, anti-rabies, anti-snake venompolyclonals, and in some instances, work more effectively than acomparable monoclonal, e.g., Sangstat's rabbit polyclonal againstthymocytes versus SIMULECT®. Drawbacks for the use of polyclonalantibodies are the risk of infectious agents (viruses, prions, bacteria)in these often pooled preparations, but also the variability inefficacy, the limited availability, the immune response directed to thepreparation if the polyclonal is non-human, and the abundance ofnon-relevant antibodies in these preparations. Polyclonals have alsobeen made using recombinant methods, but again, the production of largearrays of antibodies from individual cell lines each making oneantibody, is not economical with present biotechnological productionmethods. The production of the polyclonal antibody mixture bycultivating the many different cell lines in batch would be even moreaffected by differences in stability, growth and production rate,differences in purification yield, etc.

Provided are methods for producing mixtures of antibodies, for example,by expression from a single host cell, using antibodies with variableregions that appropriately pair with one another to yield essentiallysolely functional binding site combinations. The methods to obtain suchantibodies were described herein earlier. The resulting variable regionscan thus be co-expressed in biotechnologically viable and simpleprocedure, and a mixture of antibodies isolated using methods known inthe art.

After selection of antibodies with the appropriate pairing behavior(such as antibodies with pairing-compatible variable regions,co-expression-compatible elements, etc., as described above), theantibody variable region genes are cloned into expression vectors thatwill direct the expression of an antigen binding fragment in, forexample, the following format: Fab, Fab′, Fab′2, IgG, IgM. In manyinstances the use of antibodies with, for example, pairing-compatiblevariable regions simplifies the DNA constructs that mediate theexpression of the particular antibody format. For example, for theexpression of two different antibodies as Fab′2 fragments in which oneof the two antibody chains is the pairing-compatible variable region,only three antibody chains instead of the normal four have to beexpressed to form two different binding sites. Such simplifiedexpression constructs can lead to a more stable and more readilycontrolled expression system, and increase functional yields byminimizing problems associated with mispairing of heavy and light chaindomains.

The mixture may contain a given selection of antibodies, recognizingepitopes on the same or different targets; examples are given below. Anew application is the use of mixture containing antibodies specific forcomplexes formed by another antibody bound to a given target. Both ofthe antibodies can be provided in the mixture, providing a firstantibody to bind the antigen, and a second one to “seal” the firstinteraction, providing the antibody mixture with an increase in overallaffinity and specificity. Another embodiment is to use asymmetricallypaired antibody molecules in the mixture such that the effectorfunctions of the resulting mix are altered. The purpose of such mixingis to alter the properties of the effector mechanism of the individualantibodies in the mixture, in an antigen-specific / binding sitedirected manner, for example, the monospecific antibodies may each havea different effector from the bispecific components present in themixture. Consider the next example, a mixture of two antibody bindingsites formatted as OLIGOCLONICS® in the IgG-format, composed of theheavy chain gamma-1 heavy chain for one antibody variable region and thegamma-4 heavy chain for the other antibody variable region. TheOLIGOCLONICS® mixture will contain the two monospecific antibodies,which will be either an IgG1 or an IgG4 isotype and display theirrespective effector functions, and also a hybrid dimer of gamma-1 andgamma-4, with altered or lost effector functions. Since many Fcreceptors bind in an asymmetric manner to the symmetrically arranged Fcregion, asymmetric Fc regions often will loose interactions with Fcreceptors and thus ADCC or other activity. Mutants of Fc regions with,for example, mutations in the Fcgamma-Receptor motif (residues 233-238in the CH2-lower hinge region), or mutants with reduced C1q binding, ormutants with exchanged or shortened hinge, or with domains exchanged byother domains of the immunoglobulin heavy chain family, or Fc regionsoptimized for their interaction with particular Fc regions (e.g.,improved binding to the activating receptor FcgammaRIII and/or decreasedbinding to the inhibitory receptor FcgammaRllb), can also be used forthe assembly of such asymmetric Fc regions. Applications of suchasymmetric pairs are provided in a mixture of one compound but notothers with a particular effector function, or to remove an effector,for example, in the bispecific or monospecific compounds.

10. Examples of Uses of Compositions of Multiple Proteinaceous Compoundswith Different Binding Specificities

There are applications for mixtures of different binding sites on thesame antigen, for mixtures of different binding sites on differentantigens, for mixtures of different binding sites on different antigenson the same or different target. As an example of use of a mixture inthe treatment of a viral disease, the example of hepatitis B virus (HBV)infection is discussed. Recombinant HBV vaccines provide a safe andeffective means for prevention of HBV conferring long-term immunitythrough active immunization. In contrast to the slow onset of protectionfollowing this vaccination, passive immunotherapy with antibodies to HBVprovides immediate but short-term protection against viral transmissionand infection. Antibodies are believed to inhibit infection by blockingHBV from entering into cells. Such passive immunotherapy is advisablefor individuals who were exposed to HBV-positive material (needle or cutinjuries) and for newborns to mothers who are HBV carriers, for patientsundergoing liver transplantation. At present, such treatment is carriedout with hepatitis B immunoglobulin, a plasma derived, polyclonalantibody preparation obtained from donors who were anti-hepatitis Bsurface antigen antibody-positive. The availability of this serum islimited and further pricing and safety concerns regarding the use ofblood products, make the development of an alternative treatmentnecessary. A human monoclonal antibody would be advantageous bypresenting a stable and reproducible source for prolonged immunotherapy.However, studies show that a monoclonal antibody directed to the Santigen and neutralizing capacity against HBV in chimpanzees delayed butnot prevented the infection with HBV. In part this may be caused by theemergence of escape variants, mutants in the S-antigen that can nolonger be bound by the monoclonal antibody. Similarly, escape mutantsarise in patients after liver transplantation in clinical trials withmonoclonal antibodies. Therefore, treatment with a single monoclonalantibody may be inefficacious and insufficient. Follow-up studies haveinvolved mixtures of human monoclonal antibodies. Studies carried out byXTL Biopharmaceuticals and colleagues show that a mixture of twoantibodies is more effective in reducing viral load and inhibiting HBVinfection in animal model systems than a polyclonal mixture. Thisindicates that the potency of a polyclonal humoral immune response canbe deconvoluted to a few antibodies, and that a defined mixture of a fewantibodies should work as well or better than some polyclonalpreparations. A mixture of two antibodies recognizing different epitopeson the viral surface was thus shown to be more effective in theprevention of HBV reinfection.

In another example of use of a mixture of monoclonal antibodies in thetreatment of a viral disease, the example of the Human ImmunodeficiencyVirus type-1 (HIV-1) is discussed. Infection with HIV-1 leads to thedevelopment of the Acquired Immunodeficiency Syndrome (AIDS) if leftuntreated. During infection with HIV-1, neutralizing antibodies that aredirected against diverse epitopes on the HIV-1 envelope glycoproteinmolecules gp41 and gp120 develop. In a clinical trial published in 1992,the administration of HIV-1 seropositive plasma containing high titersof HIV-neutralizing antibodies, was associated with a reduction in HIV-1viremia and a number of opportunistic infections. Several groups havesubsequently published that administration of HIV-1 seropositive plasmaresults in delay of the first AIDS-defining event and improvement ofclinical symptoms. However, enthusiasm for passive immunotherapydeclined when it was found that antibodies failed to eliminate the virusand resulted in the emergence of neutralization escape variants inpatients. It was demonstrated that the antibodies that are inducedduring natural HIV-1 infection poorly neutralize the virus, resulting ina low potency of hyperimmune sera used for passive immunotherapy ofHIV-1 infection. In addition, it was demonstrated that some antibodiesthat arise during natural infection can even enhance the infection. Itwas realized that for antibody therapy of HIV-1, potent andwell-characterized neutralizing monoclonal antibodies were needed.

These early findings spurred the development of human monoclonalantibodies against HIV-1 envelope glycoproteins. In recent years, anumber of human monoclonal antibodies against the HIV-1 gp41 and gp120viral coat glycoproteins have been isolated and characterized for theirvirus-neutralizing activity in vitro. Subsequent experiments innon-human primate models of HIV infection and transmission have shownthat human monoclonal antibodies targeting different HIV-1 envelopeglycoprotein epitopes exhibit strong synergy when used in combination.It has been suggested that combinations of human anti-HIV monoclonalantibodies can be exploited clinically for passive immunoprophylaxisagainst HIV-1.

A third example relates to the rabies field. Rabies is an acute,neurological disease caused by the infection of the central nervoussystem with rabies virus, a member of the Lyssavirus genus of the familyof Rhabdoviridae. Almost invariably fatal once clinical symptoms appear,rabies virus continues to be an important threat to human and veterinaryinfection because of the extensive reservoirs in diverse species ofwildlife. Throughout the world, distinct variants of rabies virus areendemic, in particular, terrestrial animal species, with relativelylittle in common between them. Rabies virus is characteristicallybullet-shaped, enveloped virion of single-stranded-negative sense RNAgenome and five structural proteins. Of these, a suitable target forneutralization is the viral glycoprotein (G). Antigenic determinants onG vary substantially among the rabies virus strains. Prompt treatmentafter infection consists of passive and active immunotherapy. Forpassive immunotherapy mostly pooled serum of rabies immune individualsor immunized horses is used, but with a risk of contamination with knownor unknown human pathogens, or the risk of anaphylactic reactions,respectively. In addition, anti-rabies immunoglobulin is expensive andmay be either in short supply or non-existent in most developingcountries where canine rabies is endemic. There is, therefore, a needfor compositions and methods for producing mixes of antibodies, forexample human antibodies, to use in passive immunotherapy of rabiesinfections. A number of human monoclonal antibodies made by fusion ofEpstein-Barr Virus transformed, rabies-virus-specific humanheterohybridomas have been made (Champion et al., J. Immunol. Methods(2000) 235:81-90). A number of virus-neutralizing antibodies derivedfrom these antibodies have also been cloned (PCT/IS02/26584 andPCR/US01/14468 and Morimoto et al. (2001), J. Immunol. Methods252:199-206). Several other rabies-neutralizing monoclonal antibodieshave been described in the art, which could also be used in theexperiments below. As indicated in these publications, a mix ofdifferent rabies-neutralizing human antibodies would be an ideal reagentfor passive immunotherapy of rabies.

In general for viral diseases, the functional assembly of mixes ofanti-viral antibodies may increase the clinical efficacy of thetreatment when compared to monoclonal therapy, by decreasing theprobability of viral escape mutants resistant to treatment, and byreducing the likelihood of viral resistance with prolonged therapy. Inthe mixture, antibodies may be included that bind to many differentepitopes of the virus. It may also be feasible to include antibodies todifferent subtypes of the virus, to broaden the utility of the drug fora wider patient population. Further anti-viral antibodies directed tolinear epitopes may be added, which may be less prone to the effect ofescape mutants than conformation-dependent antibodies. The effect ofmultiple binding specificities present in the antibody mix can provide astronger signal for viral clearance than when a monoclonal antibody isused. There are also applications for mixtures of essentially onebinding site with different fine-specificities for binding its antigen.For example, when the antigen is prone to mutation as is the case withmany viral antigens, in the course of a treatment the epitope on theantigen may be altered such that the binding of the original antibody islost. When using a mixture, e.g., based on the same heavy chain pairedwith a small set of light chains that provide a range offine-specificities, there is a possibility that the mutations willaffect the binding of some species in the mixture, but not of otherswith a different binding chemistry mediated by the pairing-compatiblevariable region. In such a case, distinct binding chemistries for theinteraction with the antigen may be used, thus the pairing-compatiblevariable regions should be as unrelated as possible in sequence.Alternatively, antibodies can be used that use very different bindingsite chemistries by having unrelated heavy and light chain variableregions, but display exclusively pairing behavior such that theirproduction in the same cell yields mainly functional binding sites. Suchmixtures are may be more active than the individual components, and insome case will act synergistically.

In the OLIGOCLONICS® format, antibodies of the IgG isotype are made byco-expression of the light and heavy chain genes with appropriatepairing behavior in the same host cell. The result of this process is amixture of different proteins, the monospecific bivalent antibodieswhich carry two identical binding sites, and bispecific antibodies,carrying two different binding sites. There will be occasions where thepresence of this bispecific antibody class will further enhance theefficacy of the antibody mixture. Only when there are multiple epitopespresent on the antigen or microorganism, and these epitopes arepresented in the correct positioning, will a monoclonal antibody of theIgG isotype, for example, be able to bind both of its binding Fab-armsto the antigen. In many instances where the antigen is a monomer or asmall multimer, like cytokines, interleukins and interferons, mostlyonly one arm of a monoclonal IgG antibody will be binding the antigen.The bispecific component of the OLIGOCLONICS®, provides a newopportunity to bridge neighboring epitopes, and form a highly avidbinding antibody. Pairs that have this behavior may be selected usingthe methodologies of screening mixtures of antibodies as disclosedherein. Besides this avidity advantage, bispecific molecules may alsocross-link receptors that mono-specific yet bivalent antibodies in thesame mixture cannot cross-link. OLIGOCLONICS® may thus provide anantibody mixture that per unit of mass will more effectively neutralizeviruses, cytokines, toxins etc when compared to monoclonal antibodies,and in specific cases, for example, with an avidly binding bispecificcomponent or receptor-cross-linking or other unique mechanism mediatedby the bispecific antibody, also compared to mixtures of monoclonalantibodies. The bispecific compounds are also useful to explore routestraditionally developed with bispecific antibodies, such as theretargeting of immune effector molecules or cells such as T-cells,complement proteins and Fc-receptor expressing cells to tumor cells orpathogens.

Thus, mixtures of antibodies may be suitable to fight pathogensincluding viruses like HIV and rabies, bacteria, fungi, and parasites.Other examples where a polyclonal serum or gammaglobulin is currentlyused that could be replaced with a defined antibody mixture, includesuch diseases as rabies, hepatitis, varicella-zoster virus, herpes orrubella. Bacterial diseases that could be treated with antibody mixturesinclude Meningitis, diseases caused by Staphylococcus, Streptococcus,Hemophilus, Nesseria, Pseudomonas and the actinomycetes. Targets mayalso include those involved in bacterial sepsis such aslipopolysaccharide (LPS), lipid A, tumor necrosis factor alpha orLPS-binding proteins. Some of these pathogens occur in multipleserotypes and not one but multiple antibodies are required to neutralizethe various serotypes. A mixture of antibodies will provide, by thechoice of the binding specificities, a wider coverage of serotypes thatmay be treated and, therefore, more patients can be treated with thesame antibody mixture. The mixtures for this and other reason can formalso suitable diagnostics and part of diagnostic kits for the detectionof a disease or disorder in patient.

Mixtures of antibodies may be more effective than monoclonal antibodiesalso in the treatment of oncological diseases such as non-Hodgkin'slymphoma (NHL) and epithelial cell tumors like breast and coloncarcinoma. Targeting both CD20 and CD22 on NHL with antibodies hasalready been proven to be more effective than targeting the tumor cellswith the individual antibodies. Suitable target antigens for antibodymixtures in oncological diseases are many, including CD19, CD20, CD22,CD25 (IL-2 receptor), CD33, the IL-4 receptor, EGF-receptor, mutant EGFreceptor, Carcino-Embryonic Antigen, Prostate-specificAntigen,ErbB2IHER2, Lewis^(y) carbohydrate, Mesothelin, Mucin-1, the transferrinreceptor, Prostate-specific Membrane Antigen, VEGF and receptors, EpCAMand CTLA-4. Synergistic effects may be seen when using antibodies thatbind different targets and pathways in the disease, such as antibodieswith anti-angiogenesis and anti-proliferative effects. There are alsoapplications in this field for a mixture of essentially one binding sitewith different affinities for binding its antigen. For example, theefficiency of in vivo solid tumor penetration is limited for highaffinity antibodies due to the binding site barrier, yet a minimalaffinity is required to achieve a substantial accumulation in the tumor.With the methods described in this document, a mixture of antibodies canbe established, e.g., based on the same heavy chain paired with a smallset of light chains yet appropriate pairing behavior that provide arange of affinities when paired with the heavy chain Such mixtures canbe used to increase the accumulation in the tumor, and the best balancedcocktail found by choosing the components and their expression levels.Such mixtures may be more active than the individual components, and mayact synergistically.

Mixtures of antibodies may also be suitable to neutralize multipledifferent targets, for example, in the field of inflammatory diseases,where multiple factors are involved one way or another in mediating thedisease or aggravating its symptoms. Examples of these diseases arerheumatoid arthritis, Crohn's disease, multiple sclerosis,insulin-dependent diabetes, mellitus and psoriasis. Optimal treatment ofmany of these diseases involves the neutralization or inhibition ofcirculating pathological agents and/or those on the surface on cellstargeted in the specific inflammatory response in the patient. Inautoimmunity and inflammatory diseases suitable targets are generallyinterferons, cytokines, interleukins, chemokines and specific markers oncells of the immune system, and, in particular, alpha interferon, alphainterferon receptor, gamma interferon, gamma interferon receptor, tumornecrosis factor alpha, tumor necrosis factor receptor, HLA-class IIantigen receptor, interleukin-lbeta, interleukin-lbeta receptor,interleukin-6, interleukin-6 receptor, interleukin-15, interleukin-15receptor, IgE or its receptor, CD4, CD2, and ICAM-1.

Mixtures are also suitable for the neutralization of effects mediated byagents of biological warfare, including toxins such as Clostridiumbotulinum-derived botulinum neurotoxin, anthrax, smallpox, hemorrhagicfever viruses, and the plague. The neutralization of the botulinumtoxins is discussed here as an example. The botulinum toxins, the mostpoisonous substances known, cause the paralytic human disease botulismand are one of the high-risk threat agents of bioterrorism.Toxin-neutralizing antibody can be used for pre- or post-exposureprophylaxis or for treatment. Small quantities of both equine antitoxinand human botulinum immune globulin exist and are currently used totreat adult and infant botulism. Recombinant monoclonal antibody couldprovide an unlimited supply of antitoxin free of infectious disease riskand not requiring human donors for plasmapheresis. A panel of human andmurine monoclonal antibodies was generated from the B lymphocytes ofhyperimmune donors and immunized mice using phage antibody displaytechnology. Single monoclonal antibodies and combinations were testedfor their capacity to protect mice from lethal doses of neurotoxin (A.Nowakowski et al. (2002) PNAS 99:11346-11350.). Whereas singlemonoclonal antibodies showed no significant protection of the miceagainst lethal doses of toxin, combinations of only three monoclonalantibodies against different epitopes on the toxin gave very potentprotection. The combination of three monoclonal antibodies neutralized450,000 lethal doses of botulinum toxin, a potency 90 times greater thenhuman hyperimmune globulin. Importantly, the potency of the monoclonalantibody mixture was primarily due to a large increase in functionalantibody-binding affinity. Thus, methods that allow the cost-effective,controlled and efficient production of mixtures of monoclonal antibodiesagainst botulinum neurotoxin provide a route to the treatment andprevention of botulism and other pathogens and biologic threat agents.As shown in this study, a mix of three antibodies that boundnon-overlapping epitopes on botulinum neurotoxin, had a synergisticeffect on toxin neutralization due to a increased overall avidity.

Mixtures of antibodies may be further applied to delay the onset ofanti-idiotype responses in patients, by providing multiple idiotypes ofan antibody family, all binding to the same target, in the simplest formamino acid mutants of the same antibody with a resulting similar bindingspecificity and affinity, to a more complex mixture of multipleantibodies directed to the same epitope.

Mixtures of antibodies can also be applied to develop derivatives of theprotein mixtures, including immunotoxins, immunoliposomes, radio-isotopelabeled versions, immunoconjugates, antibody-enzyme conjugates forprodrug-therapy (ADEPT), and immunopolymers (Allen, (2002) Nat. Rev.Cancer 2:750-783). The mixes of the antibodies can either be modified inbatch with the appropriate substances, or may be genetically fused to atoxin or enzyme encoding gene as described in the art for monoclonalantibodies.

Having generally described the embodiments, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Description of the Hybridoma-Derived Anti-RabiesAntibodies used in the Studies

This Example describes a number of rabies-neutralizing antibodies thatare used in the further examples. The following antibodies arevirus-neutralizing human antibodies: (1) JB.1 (abbreviated to JB in thenext section), described in Champion et al., J. Immunol. Methods (2000)235:81-90, and the cloning and sequence in PCT/IS02/26584; (2) JA-3.3A5(abbreviated to JA in the next section), described in Champion et al.,J. Immunol. Methods (2000) 235:81-90, the cloning in Morimoto et al.(2001), J. Immunol. Methods 252:199-206 and also in PCT/US01/14468; (3)M57, antibody and its cloning were described in Cheung et al. (1992), J.Virol. 66:6714-6720, and further in PCT/IS02/26584. The nucleotidesequences of the full heavy and light chain nucleotide sequences andalso amino acid sequences of their variable regions are disclosed in thesequence listings (SEQ ID NOS:103-114 of the incorporated SequenceListing). On the basis of the data in the literature these antibodiesall neutralize a variety or rabies isolates, but not all the same,providing a broader spectrum of neutralized isolates than when using amonoclonal.

Example 2 Production of Mixtures of scFv Antibody Fragments Based onRecloned Hybridoma-Derived Anti-Rabies Antibodies and Co-Expression

This Example describes the production of a mixture of three bindingsites as three proteins. Using as a template, the variable region genesof the three antibodies described in Example 1, cloning is used toconstruct three single-chain Fv expression cassettes, one for each ofthe antibodies, and to clone these in an appropriate expression vector.

First, the variable region genes are amplified with oligonucleotidesthat hybridize to the 5′ and 3′ ends of the nucleotide sequences andprovide appropriate restriction enzyme sites for cloning. Standardcloning techniques are described in Sambrook et al., Molecular cloning,second edition, Cold Spring Harbor Laboratory Press (1987). Clonedvariable regions genes are amplified by the polymerase chain reactionusing methods well known in the art. For antibody JA, the followingprocedure is used: primers are designed in the FR1 region and in the FR4region of the variable heavy chain nucleotide sequence, such that thevariable region is cloned downstream of a bacterial leader sequence andupstream of a continuation of the reading frame with a Gly-Ser encodingsequence. The polylinker into which the variable region heavy and lightchains are cloned is indicated in FIG. 13. The primers are designed tomaintain the amino-terminal sequence of the FR1 and FR4 regions, and toinclude a unique restriction enzyme site for cloning of the variableregion into the polylinker region of pSCFV (FIG. 13). pSCFV is a pUC119derivate which is essentially pHEN 1 (Hoogenboom et al. (1991) Nucl.Acids Res. 19:4133-4137) into which the SfiI-Nod fragment is replacedwith the SfiI-Nod sequence depicted in FIG. 13, and in which the Nodsite is followed by a c-myc tag, for detection and purification of theantibody fragment. Also the geneIII of filamentous phage is deleted inthis plasmid. Several options for directional cloning are feasible,indicated by the restriction sites locations on the polylinker map onFIG. 13. For the VH of JA, the following oligonucleotides are used toamplify the VH regions: 5′-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCAGAG GTG CAG CTG TTG GAG TCT GGG GG-3′ (SEQ ID NO:120) and the reversecomplement of 5′-ACC CGG GTC ACC GTC TCC TCC-3′ (SEQ ID NO:121). The PCRreaction is carried out with the template antibody gene which wasalready cloned, plasmid SPBN-H (Morimoto et al. (2001), J. Immunol.Methods 252:199-206), for 25 cycles, denaturation at 94° C. for 30seconds, annealing at 50° C. for 60 seconds, and elongation at 72° C.for 90 seconds, using Taq DNA polymerase (Promega, Madison, Wis.). Theresulting product of approximately 400 bp is purified, digested with therestriction enzymes SfiI and BstEII, and cloned into pSCFV, resulting inpJA-VH. Similarly, the light chain of JA is amplified from pSPBN-L withappropriately designed oligonucleotides and is cloned into pJA-VH, toyield pSCFV-JA. The integrity of the sequences is confirmed by using theAmpliTaqs cycle sequencing kit (Perkin-Elmer, Foster City, US) withspecific primers based in the vector backbone just adjacent to thevariable region inserts. Similarly, the antibody variable regions fromhybridomas JB and M57 are cloned into the single-chain Fv format.

The expression of the individual antibody fragments is done as follows.Soluble scFv fragments are expressed upon induction withisopropyl-β-D-thiogalactopyranoside (IPTG) from the lacZ promoter thatdrives the expression of the scFv in pSCFV-based plasmids, and harvestedfrom the periplasmic space of E. coli TG1 cells. To confirm binding ofthe individual scFvs, an ELISA is performed using Polysorb plates (Nunc)coated with 5 micrograms/ml of rabies virus glycoprotein. Viruspurification and glycoprotein purification have been described elsewhere(Dietzschold et al. (1996) Laboratory Techniques in Rabies, Eds Meslin,Kaplan and Korpowski), World Health Organization, Geneva, p. 175).Alternatively, a source of recombinant Rabies Glycoprotein (G) of theappropriate type is used for the coating. The sequence of rabies G isavailable to the person skilled in the art and so are cloning,expression and purification techniques.

In the next step, the scFv expression cassettes are cloned one afteranother in plasmid pSCFV-3 (depicted in FIG. 14A), which is a derivativeof pSCFV carrying unique restriction sites for cloning scFv genes, twobehind the same lacZ promoter and separated via a new ribosome-bindingsite (rbs) and signal sequence (S), and one behind anarabinose-inducible promoter, rbs and S (FIG. 14A). It also carriesdifferent tags, one for each of the scFv cassettes, c-myc (as in pSCFV;sequence EQKLISEEDL (SEQ ID NO:122)), the VSV-tag (the sequenceYTDIEMNRLGK (SEQ ID NO:123)) and the influenza Hemagglutinin (HA)-tag(the sequence YPYDVPDYA (SEQ ID NO:124)), and all followed by a stretchof three alanines and five histidines. This set-up provides a method fordetection of the individual antibodies in the mix, and a generic methodfor purification, based on immobilized metal affinity chromatography(IMAC) using methods well known in the art. The plasmid is also used inExample 17 (with restriction inserts and cloning sites described in SEQID NOS:118 and 119). The scFv genes are amplified with oligonucleotidesthat introduce the appropriate sites, and cloned into this plasmid. Thefinally resulting plasmid, pSCFV-JA-JB-M57 (FIG. 14B) is introduced intoE. coli host TG1 cell, and expression of the scFvs induced with IPTG(for JA and JB) and/or arabinose (M57). By induction with IPTG, theexpression of a mixture of two functional scFv fragments is achieved, inwhich the direct linkage favors the pairing between the intramolecularlylinked variable regions. By further induction with arabinose, anadditional scFv fragment is co-expressed. Alternatively, the three scFvexpression cassettes are cloned in separate plasmids into compatibleplasmids such as pBR322 and pACYC and maintained in the same host cellbefore induction. The binding of the mixture to rabies glycoprotein (G)is tested as before using ELISA. The contribution to the binding in themix of each of the scFv fragments is confirmed using one of threeanti-tag antibodies (the mouse monoclonal antibody 9E10 binding to humanc-Myc epitope tag (product code from abcam, www.abcam.com: ab32), andpolyclonal antibodies to the HA-tag (ab3413) or VSV-tag (ab3556). Toverify whether the production is carried out by one bacterium and itsprogeny and not by three clones that each produce one of the antibodyfragments, the culture is colony-purified after four hours in theinduction phase and the production tested of three independent clones,confirming that the expression is clonal. To determine the percentage ofcorrectly paired variable regions, the scFv mixture is first purifiedfrom the E. coli periplasmic extract using IMAC. Briefly, an IPTG andarabinose-induced 500 ml culture (kept for four hours at 30° C.), isspun at 4600×g for 20 minutes at 4° C., and the bacterial pelletresuspended in phosphate buffered saline (PBS) containing proteaseinhibitors (phenyl-methyl-sulfonyl fluoride and benzamidin). Thesolution is sonicated at 24° C. using an ultrasonic desintegrator (MSEScientific Instruments), and the suspension centrifuged at 50,000×g for30 minutes at 4° C. The supernatant fraction is incubated with TALON™resin according to the instructions of the manufacturer (Clontech).After extensive washing, proteins are eluted using 100 mM imidazole.Following this procedure, scFv fragments are further purified by gelfiltration using a Superdex 75 column (Amersham Pharmacia Biotech)connected to a Biologic instrument (Biorad). ScFv concentrations arequantitated using the bicinchoninic acid kit (Pierce). A fraction of theantibody mix is bound to a molar excess of biotinylated G protein in a0.5 ml volume. The protein with bound scFvs is captured onto the surfaceof an excess of paramagnetic Streptavidin-coated beads (200 microlitersof DYNAbeads, Dynal, Norway), in a way similar to what is described inExample 4 for phage selections. The supernatants of the mixture are thentested for the presence of scFv fragments in an SDS PAGE followed byWestern blot analysis with the anti-tag antibodies to characterize thenon-functional antibodies. The experiment provides evidence for thesimultaneous production of three scFv fragments by the same host cell,and the efficient recovery of functional binding sites, thus correctlypaired variable regions from this preparation.

Example 3 Production of Mixtures of scFv-Fc Antibodies Based on ReclonedHybridoma-Derived Anti-Rabies Antibodies and Co-Expression in aEukaryotic System

This example describes the production of a mixture of three or sixdifferent proteins composed of variable regions paired to form two orthree binding specificities. In a further example, the scFv genes aresubcloned into a eukaryotic expression vector based on pCDNA3 whichcarries the human gamma-1 region. This plasmid, VHExpress, wasextensively engineered to remove internal restriction enzyme sites(Persic et al. (1997) 187:9-18), and contains a promoter (CMV instead ofEF-1alpha as in publication), a eukaryotic leader sequence, a polylinkerwith cloning sites for an antibody variable region, the human gamma-1gene and the bovine growth hormone poly A site (FIG. 15). Further itcontains the genes encoding amp and neo resistance, and the SV40 originof replication. The full sequence is given in SEQ ID NO:115. This vectoris suitable for the expression of antibody variable region genesformatted as scFv fragments. The cloning of the scFv gene of antibody JAis carried out as follows. The scFv is used as a template in a PCRreaction with oligonucleotides 5′-TATC CGC GCG CAC TCC GAG GTG CAG CTGTTG GAG TCT GGG GG-3′ (SEQ ID NO:125) and the reverse complement of5′-ACC CGG GTC ACC GTC TCC TCC GGT GAG TCC TAG CGC TTT TCG T-3′ (SEQ IDNO:126). The PCR fragment of approximately 750-800 bp is isolated,digested with BssHII and Eco47III and cloned into similarly cut plasmidVHExpress. Similarly, the scFv genes of antibodies JB and M57 are clonedinto this plasmid; to avoid digestion at internal sites the othersuitable site is used (Bpu1102I) or a three-way ligation which alsoyields the same plasmid. The resulting plasmids with correctly clonedscFv, called respectively pscFv-Fc-JA, pscFv-Fc-JB and pscFv-Fc-M57, areintroduced into host cells, in this example PER.C6® cells.

For an initial analysis, these plasmids are transiently expressed eitheralone or in combinations of two or three scFv-Fc constructs. Cells grownto 5×10⁶ cells/ml in culture medium with 10% Fetal calf serum (FCS) in80 cm² flasks are transfected for four hours using lipofectamine(Invitrogen Life Technologies) according to the manufacturer'sinstructions (140 microliters Lipofectamine per 10 micrograms of DNA perflask) in serum-free medium at 37° C. After this incubation, cells arewashed, resuspended in rich culture medium, and the cells grown for fivedays. The supernatant is harvested for analysis of the secreted scFv-Fcfusion protein. A sandwich ELISA is used to quantify the amount of IgGproduced, using two antibodies directed to the Fc region. The scFv-Fcfusion proteins are purified using protein A affinity chromatographyusing a HighTrap column (Amersham Pharmacia) according to themanufacturer's instructions for IgG1), and the eluate concentrated viaMicrocon-YM30 concentrator (Amicon) and its buffer exchanged for PBS pH7.0. The occurrence of different scFv-Fc mixtures, six in total for thecells transfected with the three scFv-Fc genes, are furthercharacterized as described above in ELISA, and using viral isolates thatare specifically recognized by the antibodies, including European batvirus 2 for antibody JB and Lagos bat virus and Mokoa virus for antibodyJA, and strains CVS-11, CVS-24, PM, SHBRV and COSRV (Champion et al., J.Immunol. Methods (2000) 235:81-90). The presence of the M57 and JBbinding sites is confirmed using an anti-Id antibody (see, also Examples14 and 22). Following this, the viral neutralization activity of themixture of three monospecific and three bispecific molecules (withoutpurification) is assayed for the presence of rabies virus-neutralizingantibodies using the rapid fluorescent focus inhibition test (RFFIT) asdescribed by Hooper et al., ASM Pres, WA, p. 1997. Essentially, serialdilutions are made of the supernatant containing the antibody mixture in96-well plates (Nunc), and a rabies virus dilution known to cause 70-80%infection of indicator cells added to each well. Controls are positiverabies-immune serum control samples and negative medium are alsoincluded. After one hour, to each well, 50,000 baby hamster kidney (BHK)cells are added and the culture incubated overnight at 37° C. Plates arethen washed once with ice-cold PBS and the cells fixed with ice-cold 90%acetone for 20 minutes at −20° C. Acetone is removed and to theair-dried plates 50 microliters of FITC-labeled anti-rabiesnucleoprotein monoclonal antibody (ab 1002 from abcam site or antibodyfrom Centocor, Malvern) is added. After one hour incubation at 37° C.,the plates are washed three times with water and analyzed under afluorescence microscope. The activity of each of the scFv-components isstudied by testing in this assay the neutralization of a variety ofdifferent rabies isolates, including the ones mentioned in Example 1.

The same plasmids, pscFv-Fc-JA, pscFv-Fc-JB and pscFv-Fc-M57, are alsosuitable for making stable transfectants. By selection using theneo-resistance gene and culturing and screening methods known to thosein the art, stable PER.C6® derived cell lines expressing antibodies areobtained. Essentially 5×10⁶ PER.C6® cells are transfected usingLipofectamine according to the manufacturer's instructions, and 3micrograms of DNA per plasmid. Cells are transfected with the 3micrograms of each plasmid separately, or with 1.5 micrograms each ofpscFv-Fc-JA and pscFv-Fc-JB, or with 1.5 micrograms each of pscFv-Fc-JBand pscFv-Fc-M57, or with 1 microgram of each of pscFv-Fc-JA,pscFv-Fc-JB and pscFv-Fc-M57, or with a control LacZ vector. After fivehours, the cells are washed and the medium is exchanged withnon-selective medium. The next day the medium is replaced with freshmedium containing 500 micrograms/ml G418 (Sigma-Aldrich) and also everynext two to three days, the culture medium is refreshed until clonesappear (15 to 20 days after seeding). Clones are picked and cloned outto limiting dilution conditions, such that two to three weeks later,clonal cell lines start appearing. These are expanded to larger wellsand flasks, and eventually the selective medium is omitted. Thesupernatant of these cell lines is harvested for analysis of thesecreted scFv-Fc fusion protein. As before, a sandwich ELISA (asdescribed in WO 00/63403) is used to quantify the amount of IgGproduced, using two antibodies directed to the Fc region. The scFv-Fcfusion proteins are purified using protein A affinity chromatographyusing a HighTrap column (Amersham Pharmacia) according to themanufacturer's instructions for IgG1. Purified scFv-Ig from variousclones is isolated, purified and tested in a series of assays. The firstis to analyze the presence of the two or three different scFv genes ofthe cell lines created, by amplifying the genomic DNA of these celllines with antibody JA/JB or M57 scFv orV-gene-specificoligonucleotides, and confirming the presence bysequencing the amplified material. The copy number of each of theintegrated antibody constructs is determined with methods such asSouthern blot or Fluorescent In Situ Hybridization (FISH). Second, themixture is biochemically characterized using SDS-PAGE and iso-electricfocusing. Alternatively, anti-idiotype antibodies or peptide mimitopesare used to delineate the compositions (see, Example 12). The stabilityof the expression level, of the ratios between the different scFvcomponents, and of the composition of the antibody mixture produced bycell lines which produce the mix of three or six proteins is followedover time by these assays. Finally, binding and neutralization assaysare carried out, including antigen binding in ELISA and in fluorescencemicroscopy with infected cells and tissues, and in the RFFIT virusneutralization assay as described above. The biological activity of themixture is tested against a range of rabies isolates and the activitydetermined according to the international Units of Rabies Antibodies andreferenced to WHO reference Rabies Immunoglobulin (WHO Technical SeriesReport (1994) vol 848, p. 8; and vol. 840). By testing the biologicalactivity (virus neutralization) of a series of cell lines producingvariable quantities of the three scFv-Fc fusions, the most optimalmixture is identified. The mixtures are compared to the activity ofIMMOG_(AM)® Rabies, the human immunoglobulin preparation used forpassive immunotherapy (see, alsowww.aventispasteur.com/usa/product/pdffiles/!LE3439I.PDF). The effect ofthe bispecific component is tested by comparing the neutralizationefficacy of the scFv-Fc protein mixture with the activity of comparablequantities of the (1) individual whole recombinant antibodies JA (IgG1),JB (IgG1) and M57 (IgG1), (2) mixtures of two or three of theseantibodies. Due to the discrepancy observed sometimes between in vitroand in vivo neutralization data, besides in vitro neutralization tests,it may sometimes be necessary to carry out in vivo neutralization testsusing mouse protection experiments as described in Dietzschold et al.(1992) PNAS 89:7252.

Example 4 Selection of Optimally Paired Variable Regions for TwoAntibody Variable Region Pairs by Optimizing the Light Chain VariableRegion

Antibodies M57 and JB are used in this experiment. Both have a lambdalight chain, of class I for JB and class II for M57, with homologybetween the two chains (FIG. 16). The antibody heavy chain variableregion genes of these two antibodies are cloned into vectorpFab-display, which resembles functionally pCES1 (H. J. de Haard et al.(1999) J. Biol. Chem. 274:18218-18230), and is a Fab fragment displayand expression vector. In this vector system, the variable heavy chainregion genes are cloned as VH-gene fragments; the vector supplies allFabs with a human gamma-1 CH1 gene. The Fd fragment is fused to two tagsfor purification and detection: a histidine tail for Immobilized MetalAffinity Chromatography (IMAC) and a c-myc-derived tag, followed by anamber stop codon and the minor coat protein III of filamentous phage fd.The antibody light chain is cloned as full VLCL fragment, for directedsecretion and assembly with the VHCH1 on the phage particle. Restrictionenzyme sites and the sequence of the polylinker region is indicated inFIGS. 17A and 17B. The cloning of the variable regions is carried outsimilarly as described in Example 2, with oligonucleotides to amplifythe VH region and that append appropriate restriction enzyme sites. Theresulting plasmids are designated pVH-M57 and pVH-JB, respectively.

These plasmids are used as recipients for a collection of human lambdachains derived from human donors. B lymphocytes are isolated from 2-L ofblood on a Ficoll-Pacque gradient. For RNA isolation, the cell pellet isimmediately dissolved in 50 ml 8 M guanidinium thiocyanate /0.1 M2-mercaptoethanol. Chromosomal DNA is sheared to completion by passingthrough a narrow syringe (1.2/0.5 mm gauge), and insoluble debris isremoved by low speed centrifugation (15 minutes 2,934×g at roomtemperature). RNA is pelleted by centrifugation through a CsCl-blockgradient (12 ml supernatant on a layer of 3.5 ml 5.7 M CsCl/0.1 M EDTA;in total four tubes) during 20 hours at 125,000×g at 20° C. in anSW41-rotor (Beckman) RNA is stored at −20° C. in ethanol. Random primedcDNA is prepared with 250 μg PBL RNA. RNA is heat denatured for fiveminutes at 65° C. in the presence of 20 μg random primer (Promega),subsequently buffer and DTT are added according to the suppliersinstructions (Gibco-BRL), as well as 250 μM dNTP (Pharmacia), 800 URNAsin (40 U/μl; Promega) and 2,000 U MMLV-RT (200 U/μl; Gibco-BRL) in atotal volume of 500 After two hours at 42° C., the incubation is stoppedby a phenol/chloroform extraction; cDNA is precipitated and dissolved in85 μl water. From this material, the variable region gene pools from thelight chain lambda family are amplified using 4 Vλ-specificoligonucleotides that preferentially pair to the lambda I and IIfamilies (HuV11A/B/C-BACK and HuV12-BACK as in Table below) and with twoprimers based in the constant regions (HuC12-FOR and HuC17-FOR as in theTable below, combined in each reaction), and with PCR in a volume of 50μl, using AmpliTaq polymerase (Cetus) and 500 pM of each primer for 28cycles (one minute at 94° C., one minute at 55° C. and two minutes at72° C.). All products are purified from agarose gel with the QIAex-IIextraction kit (Qiagen). As input for reamplification to introducerestriction sites, 100 to 200 ng purified DNA-fragment is used astemplate in a 100 μl reaction volume, using the oligonucleotidesappropriately extended to provide the sites for cloning, ApaLI and AscI(last six primers of following Table). This amplified material ispurified, digested with AscI and ApaLI and two samples cloned into thetwo different plasmids pVH-M57 and pVH-JB.

HuV11A-BACK 5′-CAG TCT GTG CTG ACT CAG CCA CC-3′ (SEQ ID NO: 127)HuV11B-BACK 5′-CAG TCT GTG YTG ACG CAG CCG CC-3′ (SEQ ID NO: 128)HuV11C-BACK 5′-CAG TCT GTC GTG ACG CAG CCG CC-3′ (SEQ ID NO: 129)HuV12-BACK 5′-CAR TCT GCC CTG ACT CAG CCT-3′ (SEQ ID NO: 130) HuC12-FOR5′-TGA ACA TTC TGT AGG GGC CAC TG-3′ (SEQ ID NO: 131) HuC17-FOR5′-AGA GCA TTC TGC AGG GGC CAC TG-3′ (SEQ ID NO: 132) HuV11A-BACK-APA5′-ACC GCC TC ACC AGT GCA CAG TCT GTG CTG ACT CAG CCA CC-3′(SEQ ID NO: 133) HUV11B-BACK-APA5′-ACC GCC TCC ACC AGT GCA CAG TCT GTG YTG ACG CAG CCG CC-3′(SEQ ID NO: 134) HuV11C-BACK-APA5′-ACC GCC TCC ACC AGT GCA CAG TCT GTC GTG ACG GAG CCG CC-3′(SEQ ID NO: 135) HUV12-BACK-APA5′-ACC GCC TCC ACC AGT GCA CAR TCT GCG CTG ACT CAG CCT-3′(SEQ ID NO: 136) HuC12-FOR.ASC5′-ACC GCC TCC ACC GGG CGC GCC TTA TTA TGA ACA TTC TGT AGG GGC CAC TG-3′(SEQ ID NO: 137) HuC17-FOR-ASC5-ACC GCC TCC ACC GGG CGC GCC TTA TTA AGA GCA TTC TGC AGG GGC CAC TG-3′(SEQ ID NO: 138)

This cloning results in two libraries designated as Fab-VH-M57-VLn andFab-VH-JB-VLn.

Phage particles are made from cultures of these two libraries. Therescue of phagemid particles with helper phage M13-K07 is performedaccording to (Marks et al. (1991), J. Mol. Biol. 222:581-597) on a 1-Lscale, using representative numbers of bacteria from the library forinoculation, to ensure the presence of at least ten bacteria from eachclone in the start inoculum. For selections, 10¹³ cfus (colony formingunits) are used with 10 micrograms/ml Rabies glycoprotein coated inimmunotubes (Maxisorp tubes, Nunc) or with 250 nM soluble biotinylated Gprotein. Antigen is biotinylated at a ratio of one to five moleculesNHS-Biotin (Pierce) per molecule antigen according to the supplier'srecommendations. Three rounds of selection are carried out with theselibraries. Detailed protocols for culturing and selecting phage displaylibraries have been described elsewhere (as in Marks et al. (1991), J.Mol. Biol. 222:581-597) and are well known to those working in the art.Briefly, the selection with the biotinylated antigen is carried out asfollows. Phage particles are incubated on a rotator wheel for one hourin 2% M-PBST (PBS supplied with 2% skimmed milk powder and 0.1%TWEEN®-20). Meanwhile, 100 μl Streptavidin-conjugated paramagnetic beads(Dynal, Oslo, Norway) are incubated on a rotator wheel for two hours in2% M-PBST. Biotinylated antigen is added to the pre-incubated phage andincubated on a rotator wheel for 30 minutes. Next, beads are added andthe mixture is left on the rotator wheel for 15 minutes. After 14 washeswith 2% M-PBST and one wash with PBS, phage particles are eluted with950 μl 0.1 M triethylamine for five minutes. The eluate is immediatelyneutralized by the addition of 0.5 ml Tris-HCl (pH 7.5) and is used forinfection of long-phase E. coli TG1 cells. The TG1 cells are infectedfor 30 minutes at 37° C. and are plated on 2×TY (16 g Bacto-trypton, 10g Yeast-extract and 5 g NaCl per liter) agar plates, containing 2%glucose and 100 μg/ml ampicillin. After overnight incubation at 30° C.,the colonies are scraped from the plates and used for phage rescue asdescribed (Marks et al. (1991), J. Mol. Biol. 222:581-597). Culturesupernatants of individual selected clones harboring either rescuedphage or soluble Fab fragments are tested in ELISA with directly coatedantigen or indirectly captured biotinylated antigen via immobilizedbiotinylated BSA-streptavidin. Here described is the procedure withbiotinylated antigen for the detection of soluble Fab fragments. Forcapture of biotinylated Rabies glycoprotein, first biotinylated BSA iscoated at 2 μg/ml in PBS during one hour at 37° C. After three washeswith PBS-0.1% (v/v) TWEEN®-20 (PBST), plates are incubated during onehour with streptavidin (10 μg/ml in PBS/0.5% gelatin) (24). Followingwashing as above, biotinylated antigen is added for an overnightincubation at 4° C. at a concentration of 3 μg/ml. The plates areblocked during 30 minutes at room temperature with 2% (w/v) semi-skimmedmilk powder (Marvel) in PBS. The culture supernatant is transferred tothese wells and diluted 1 or 5-fold in 2% (w/v) Marvel/PBS and incubatedfor two hours; bound Fab is detected with anti-myc antibody 9E10 (5μg/ml) recognizing the myc-peptide tag at the carboxyterminus of theheavy Fd chain, and rabbit anti-mouse-HRP conjugate (DAKO). Followingthe last incubation, staining ms performed with tetramethylbenzidine(TMB) and H₂O₂ as substrate and stopped by adding half a volume of 2 NH₂SO₄ the optical density is measured at 450 nm. Clones giving apositive signal in ELISA (over 2× the background), are further analyzedby BstNI-fingerprinting of the PCR products obtained by amplificationwith the oligonucleotides M13-reverse and geneIII-forward (as in Markset al. (1991), J. Mol. Biol. 222:581-597).

Large-scale induction of soluble Fab fragments from individual clones isperformed on a 50 ml scale in 2×TY containing 100 μg/ml ampicillin and2% glucose. After growth at 37° C. to an OD₆₀₀ of 0.9, the cells arepelleted (ten minutes at 2,934×g) and resuspended in 2xTY withampicillin and 1 mM IPTG. Bacteria are harvested after 3.5 hours growingat 30° C. by centrifugation (as before); periplasmic fractions areprepared by resuspending the cell pellet in 1 ml ice cold PBS. After 2to 16 hours rotating head-over-head at 4° C., the spheroplasts areremoved by two centrifugation steps: after spinning during ten minutesat 3,400×g, the supernatant is clarified by an additional centrifugationstep during ten minutes at 13,000×g in an Eppendorf centrifuge. Theperiplasmic fraction obtained is directly used for determination of theaffinity by surface plasmon resonance and of fine-specificity in westernblot or virus neutralization studies.

Using the cited ELISA test, panels of antigen reactive Fabs areidentified for both M57 and JB. The Fabs are purified and their relativeaffinity for the antigen compared to the native antibody as Fabdetermined. All clones that are in a ten-fold reach of the affinity aresequenced. For sequencing, plasmid DNA is prepared from 50 ml culturesgrown at 30° C. in medium, containing 100 μg/ml ampicillin and 2%glucose, using the QIAGEN midi-kit (Qiagen). Sequencing is performedwith the thermocycling kit (Amersham) with CY5-labeled primers CH1FOR(5′-GTC CTT GAC CAG GCA GCC CAG GGC-3′ (SEQ ID NO:139)) and M13REV(5′-CAG GAA ACA GCT ATG AC-3′ (SEQ ID NO:140)). The analysis is done asdescribed above: the amino acid sequences of the two antibody VL sets,for M57 and JB, are compared to one another. Many of the selectedvariants are derived from the lambda 1 and lambda 2 family but carrysomatic mutations throughout the sequence. In each collection, a set of10 VLs are selected that are putative “common” candidates for pairing toboth VHs, and these are cloned via the common restriction sites ApaLland Asci into the plasmid carrying the other VH. Thus, the VLCL of acandidate clone of library Fab-VH-M57-VLn is isolated usinggel-electrophoresis of the ApaLI-AscI digest and cloned into pVH-JB.This is carried out for all candidate VLs; the new combinations are alltested as before in ELISA for their pairing compatibility with thenon-cognate VH. The clone with highest affinity in both antibodies isdesignated VL-M57=JB. This procedure leads to the identification of alambda variable region light chain that in the Fab format can optimallypair with both the VH of JB and of M57.

Example 5 Selection of Optimally Paired Variable Regions for TwoAntibody Variable Region Pairs by Optimizing the Heavy Chain VariableRegion

For occasions where the two light chains of two given antibodies arevery different from one another, as is the case between antibodies ofkappa and lambda families, it is also possible to follow an alternativestrategy than the one described in Example 4. Herein described is theselection of an optimally paired VL that will be pairing in a compatiblefashion with two VH variable regions. In the experiment, the major loopin the VH, the CDR3 that is both responsible for antigen binding andcontributes to the interaction with the light chain, is diversified.Other schemes can be followed, in which other VH residues known to bestructurally positioned at the VH-VL interface are mutated (exemplifiedin FIGS. 18A-18F). This procedure may also be applied to multiplevariable region genes, using, for example, a chosen germ line encodedvariable region gene and multiple partner variable regions which arethen mutagenized and selected as in the following description.

The aim of the experiment is to find a JA-variant that will haveoptimally pairing behavior to VL-M57=JB. The JA antibody carries a kappachain instead of a lambda (FIG. 16), and replacement of its cognatelight chain with VL-M57-JB leads to a substantial loss of affinity.Therefore, it is the VH of this antibody that will be mutated, tocompensate for loss of affinity with the antigen, and to provide alsonew potential interactions with the new VL. First, the VL-M57=JB iscloned as VLCL ApaLI-AscI fragment into pFab-display as described inExample 4; this yields plasmid pVL-M57=JB. The heavy chain of antibodyJA is amplified from pVH-JA (Example 2) using two primers: 5′-GTC CTCGCA ACT GCG GCC CAG CCG GCC ATG GCA GAG GTG CAG CTG TTG GAG TCT GGGGG-3′ (SEQ ID NO:141), and the reverse complement of the followingsequence, which is a mutagenic oligonucleotide that is spiked withmutations in the two residues preceding the CDR3 and throughout the CDR3region (in the underlined region; see also FIGS. 18A-18F):

(SEQ ID NO: 142) 5′-C ACG GCC GTA TAT TAC TGT GCG AAA GAT CGA GAGGTT ACT ATG ATA GTT GTA CTT AAT GGA GGC TTT GACTAC TGG GGC CAG GGA ACC CGGG TCA CCG TCT CCT-3′.

The spiking is carried out by the inclusion during the oligonucleotidesynthesis at the underlined residues, of mixes of 90% of the naturalresidue, and 10% of a mix with equimolar ratios of the four residues.The PCR is carried out as in Example 1 to yield a 350-400 bp fragment,which is gel-purified, digested with SfiI and BstEII and cloned intopVL-M57=JB, to form a library of variants of JA, designatedFab-JA-YHmut.

This library is now rescued using helper phage and selections andscreenings are carried out on Rabies glycoprotein according to themethods described in Example 4. The resulting Fab clones that maintainantigen binding contain a VH-JA variant that is pairing-compatible withVL-M57=JB. Candidate Fabs are produced and purified, and their affinitydetermined as described in Example 4. The variable heavy chain mutant ofthe highest affinity is designated VH-JA*.

Example 6 Isolation of Antibodies Against Rabies Glycoprotein from aRandom Combinatorial Phage Library and Screening for Compatible VLbetween Binding Clones

Phage display libraries are a suitable source of antibodies. Librariesthat are suitable for the assembly of the panels of antibodies includenon-immune libraries (H. J. de Haard et al. (1999) J. Biol. Chem.274:18218-18230), semi-synthetic libraries (de Kruif et al. (1995) J.Mol. Biol. 248:97, and Griffiths et al. (1994) EMBO J. 13:3245-3260) andalso immune libraries, which often display a lower level of variablechain diversity. The first application presented is to select antibodiesto one antigen only, providing a mixture of antibodies directed to thesame antigen that can then be screened for pairing-compatible variableregions, and used to produce an antibody mixture. The second applicationconcerns the selection of antibodies to two different antigens. Methodsto carry out selections and screenings are well known in the art and arealso described in Examples 4 and 5. Using selection on antigens, panelsof antibody fragments specific for a given set of antigens are obtained.For each of the panels the sequence of VH and VL is determined. Thus,each antigen will have a set of reactive antibodies. It is then possibleto identify by visual inspection in each of the panels those antibodiesthat share a given VL or have highly related VLs between the differentsets. The cases described in Example 4 are also applicable here. In thebest case each set has at least one antibody with an identical VL as atleast one other antibody in the other sets. When this is not the case, asuitable VL that matches a given VH is found by the methods described inExample 4: the VH is paired with a repertoire of VLs, of which thecomposition is driven by the homology with a given VL or VLs.Alternatively, one VL is chosen and the non-matching VH is mutagenizedas described in Example 5, to yield compatible pairs for all sets. Thesequences are further inspected to find pairing-compatible variableregions that do not have sequence identity or homology. Variable heavychains that pair with multiple variable light chains and vice versa areidentified. Such “promiscuous” pairings imply that the variable regioninvolved binds to the same antigen with any of several partner chains Torapidly identify such variable regions, it is particularly useful to usesemi-synthetic antibody libraries which have a limited number ofpositions which were diversified, as has been described for the humansynthetic phage antibody library in Griffiths et al. (1994) EMBO J.13:3245-3260.

In the first application, antibodies are selected against one antigen,the Rabies glycoprotein. The library described in Griffiths et al.(1994) EMBO J. 13:3245-3260, is selected on the Rabies glycoproteinantigen as described earlier. There are different sources of theantigen, including the material purified as in Dietzschold et al. (1996)Laboratory Techniques in Rabies, Eds Meslin, Kaplan and Korpowski, WorldHealth Organization, Geneva, p. 175. Alternatively, a source ofrecombinant Rabies Glycoprotein (G) of the appropriate type is used forthe coating. The sequence of rabies G is available to persons in the artand so are cloning, expression and purification techniques. A suitableformat is to use an immuno-adhesion-type of molecules, in which thesoluble part of the glycoprotein is genetically fused to animmunoglobulin Fc region, and the fusion protein expressed in eukaryoticcells (see, also Chamow and Ashkenazi, Antibody Fusion Proteins, 1999,Wiley-Liss, N.Y.). For phage selection, the immuno-adhesion isbiotinylated to be used in a selection as described in Example 4, orimmobilized by coating. Alternatively, selections are carried out onimmobilized (or biotinylated) Rabies virions, and selections are carriedout each round on virions derived from different Rabies strains, toobtain a panel of antibodies that recognize the most common epitopespresent in the different strains. These procedures yield a panel ofantibodies directed to the Rabies antigen, but the compatibility of thepairing of variable regions of the individual candidates has to betested.

Disclose herein are the use of the antibodies from the phage antibodylibrary described by Griffiths et al. (1994) EMBO J. 13:3245-3260, butfor the clones from other libraries the same principles apply. A panelof Fabs reactive with the Rabies glycoprotein is identified and theprocedure to find optimally pairing VH and VL combinations as describedabove carried out. As an alternative, independent of sequencing, toidentify optimally paired VH and VL pairs (that, for example, are missedin the sequencing analysis), the following empirical approach isfollowed. The variable light chains of a panel of 30 human antibodiesare shuffled, and the new combinations tested in a binding assay. Theshuffling is carried out by recloning the light chains present in theantigen reactive Fab clones which are based in the recombinedfd-DOG-21ox-plasmid, as ApaLI-AscI fragment into the same Fab-containingphage genomes cut with the same (unique) enzymes. This is an experimentthat is done in batch, with all 30 VL inserts and 30 VH-containingvectors mixed; sequencing is used to delineate the pairing of each VH-VLpair. ELISA is used to define which antibodies retain antigen bindingactivity and those clones are sequenced. The resulting combinationsprovide VH-VL which are pairing-compatible, the first class of which isformed by clones that share a VL or related VL; in that case one can bechosen plus the different VH genes for making OLIGOCLONICS® (see,Example 10). The second class contains clones with “promiscuous”pairing, and the VH genes of these are combined with the VH and VL pairsof those Fabs which are compatible with this tolerant VH.

The second application concerns the selection of phage antibodies on twodifferent antigens, as indicated in FIG. 2. The same procedures as werejust described for one antigen are followed, now to assemble two sets ofantibodies, one for each antigen. The same procedures are followed alsoto identify clones with an identical or similar variable regionsequence, or empirically, to demonstrate the existence ofpairing-compatible antibodies between the two sets of antibodies.

Example 7 Isolation of Antibodies Against Rabies Glycoprotein from aPhage Library with Limited Diversity and Screening Antibodies which areNon-Competitive

Phage antibody scFv or Fab libraries that are formed by focusing thediversity in one variable region and keeping the other variable regioninvariable, for example a germ line sequence, are particularly relevant.From such libraries it is feasible to isolate antibodies with adifferent heavy chain yet identical light chain, or vice versa (FIG. 3).Such antibodies are readily reformatted into an OLIGOCLONICS® format. Inthe art, it has been described that antibodies that share the same VLgene but have different VH genes and widely varying specificities can beobtained from phage antibody display libraries (Nissim et al. (1994),EMBO J. 13:692-698).

A sub-library of the semi-synthetic scFv library (de Kruif et al. (1995)J. Mol. Biol. 248:97) is used in the following example. This sub-librarycontains antibodies with diversity in the VH region only. Selections onantigen are carried out as described in the previous examples. UsingRabies glycoprotein as the antigen as described in Example 6, ten humanantibodies with different VH yet identical VL are identified. These areimmediately suitable for inclusion into OLIGOCLONICS® (Example 10). Insome instances it will be favorable to identify those antibodies thatrecognize different epitopes from the other antibodies in the mixture,and/or to obtain antibodies that recognize the same epitope recognizedby a given monoclonal and polyclonal antibody. The competitive nature ofthe selected ten scFv antibodies with the Rabies monoclonal antibody M57is determined in ELISA, using the set-up described in Example 2(essentially, with bound antigen, adding sample, and detecting using anHRP-labeled anti-c-myc antibody) in the presence or absence of the M57antibody. Competition experiments between the clones are readilyperformed using similar competition ELISAs with the phage-scFv particlesand the soluble scFv fragments. Besides this procedure to screen clonesfor a particular competition-behavior, it is also possible to influencethe selection outcome, either by using an antibody to block a site onthe antigen during the selection (preventing antibodies to or competingwith this epitope from being selected), or by using an antibody tocompetitively elute the fraction of phage antibodies that is bound tothe same epitope. Examples of both are known in the art and methods areapplicable here also to define suitable antibody combinations forinclusion in the OLIGOCLONICS® composition.

Example 8 Isolation of Single-domain Antibodies Against RabiesGlycoprotein from a VL Phage Library, and Pairing with a SuitableVariable Region

Antibodies made in two steps are also suitable for the inclusion in theOLIGOCLONICS® format and to make antibody mixtures. Rabies-specificsingle domain VL antibody fragments are selected from a phage displayedrepertoire isolated from human PBLs and diversified by DNA-shuffling, asdescribed in van den Beucken et al. (2001), J. Mol. Biol. 591-601(libraries B and C). Selection and screening experiments are done asdescribed in the previous examples. After the third round of selection,the pool of VLs is taken for combination with one VH segment (asdepicted in FIG. 4, column (e)). For this, the VL pool is recloned byPCR as an ApaLI-XhoII fragment into pFab-display (FIGS. 17A and 17B)into which is cloned a single human VH. The latter is a DP-47 germ lineencoded variable region with short CDR3 sequence designated VH-N (SEQ IDNO:116), which is obtained by providing via PCR antibody clone FITC-B11from Table IV in Griffiths et al. (1994) EMBO J. 13:3245.3260, with ashort, five-residue CDR3 of amino acid sequence GGAVY (SEQ ID NO:143),and cloning this as SfiI-BstEII fragment into pFab-display. This CDR3 isfound in many different antibodies, and a short sequence with minimallength side chains (except for the tyrosine) is chosen to minimizeeffects on antigen binding and pairing. The resulting mini-library isscreened for those antibody Fab fragments that maintain antigen binding.The three best Rabies glycoprotein-specific VL genes are designatedVL-G1, G2 and G3. Similarly, the principles of this approach areapplicable to building antigen-specific heavy chain fragments based onthe VH domain, and providing these with a “neutral” VL, or even“neutral” partner VH.

Example 9 Selection of Antibodies with Pairing-Compatible VariableRegions by Intracellular Competition, and Expression af a Composition ofTwo or Three Fab Fragments with Pairing-Compatible Variable Regions

Selections with phage libraries are carried out using monoclonalantibodies as competitors during the formation of new phage particles.The selection biases the library selection towards variable region pairswith compatible pairing in the context of multiple variable regionsbeing expressed in the same host cell. The system relies on thesimultaneous expression of two or more Fab fragments, the variableregion of one of which is anchored onto a phage coat protein (FIG. 5).

First, the variable region genes of antibody M57 are cloned intopFab-Sol-pbr, a derivative of pFab-display (FIGS. 17A and 17B) with thesame polylinker, but no gIII, no M13 intergenic region and instead ofpUC119 the pBR322 backbone carrying the ampicillin resistance gene. Thevariable region genes of antibody JB are cloned in pFab-Sol-ACY-cat,similar in set-up as the previous one but carrying the Chloramphenicolresistance gene and based on the pACYC backbone. Both plasmids mediatethe expression of the soluble non-tagged Fab fragment under control ofthe lacZ promoter, and they are compatible with one another and can bemaintained in the same cell with antibiotic selection. Methods for thecloning have been described earlier; the sequences of these antibodiesare also included in the sequence listings below, thus it will bepossible for someone working in the art to clone these Fabs into thesepolylinkers such that upon induction with IPTG, both antibodies areexpressed in the periplasm of the culture. These two antibody Fabfragments form the competitors in this method. E. coli TG1 cellsharboring both plasmids are infected with phage harboring a library ofhuman Fab fragments, in which the heavy chain is anchored to the phagecoat and the light chain is provided as a soluble, non-anchored chain.The fd-based library from Griffiths et al. (1994) EMBO J. 13:3245-3260,which contains both VH and VL diversity is used for infection, theresulting bacteria start producing new phage particles and incorporatethe L and Fd chains expressed from this genome. Cells are grown to an ODof 1.0, the cells washed to remove produced phage, and the cellsincubated for four hours in 1 mM IPTG. During this time, competitionwill occur for pairing between the three variable heavy and lightchains, and there are many opportunities for mispairing. The phageproduced during this induction time will only recognize the nativeantigen, if the VH is tolerant to pair with any VL yet bind antigen, orwhen it exclusively pairs with the VL that is also encoded in thegenome. The phage is harvested, PEG precipitated, dissolved in PBS, andis now selected for binding to Rabies glycoprotein. Methods forselection have been described earlier. In both case the phage will beable to bind antigen, and be enriched in a selection round with antigen.The phage resulting from the selection is used to infect cells harboringthe two Fab-containing plasmids, and the cycle of induction, phagepreparation and selection is repeated. After five rounds of thisselection, the resulting Fab proteins are tested for antigen binding ina solid phage ELISA and recloned into the soluble expression vectorspFab-Sol-ACY-cata and pFab-Sol-pbr. E. colis are transfected with one ofthese plasmids and either the M57-containing vector or the JB-containingvector described above, or no additional vector. These cultures areinduced with IPTG (inducing expression of one or two Fab fragments), andthe resulting Fab fragments and Fab mixes analyzed for antigen bindingin ELISA. To confirm exclusive or tolerant pairing, the Fab fragmentsare purified using IMAC and tested in a capture assay with antigen asdescribed in Example 2. The selected variable region pair can be furtherused to build an OLIGOCLONICS® mixture with either M57 or JB variableregion genes (but not together), as in Example 10.

For making a mix of these three antibodies, the experiment is repeatedusing the VL-M57=JB from Example 4 instead of the two original lightchains VL-M57 and VL-JB. The result of the selection is a small numberof Rabies antigen-specific VH-VL pairs derived from the phage library.The best candidate according to affinity, with designated variableregions VH-POI and VL-POI, is further tested as above to confirm that itis pairing-compatible with the VH-57, the VH-JB and the VL-M57=JB. Next,the following expression cassettes are introduced in the same E. colihost cell using the two plasmids described earlier for producing thecompeting Fab, using cloning methods familiar to those working in theart: in cassette (1), on one plasmid, the VL-M57=JB-CL and VH-CH1 ofM57; in cassette (2), the VL-M57=JB and VH-CH1 of JB (a second copy isprovided to obtain an excess of light chain for pairing with the twoheavy chains); and in cassette (3), on the other plasmid, the VL-POI-CLand VH-POI-CH1. Induction with IPTG leads to the production of a mixtureof Fab fragments with paired variable regions, which is then recoveredusing IMAC purification. Alternatively, protein G purification is used.Using the binding and other assays described in the earlier examples forRabies glycoprotein antibodies, the mixture is characterized. Thecontents of the mixture is dependent on the growth and inductionconditions of the bacteria and the primary amino acid sequences of theFab genes.

Example 10 Methods for Production of OLIGOCLONICS in Eukaryotic Cells

A method for producing a mixture of antibodies in eukaryotic cells usingexpression in a recombinant host cell of multiple VH and VL genesresulting in the production of VH and VL proteins capable of pairing toform functional bivalent and bispecific antibodies, named OLIGOCLONICS®,is exemplified herein. The general format of a eukaryotic expressionvector for human monoclonal antibodies is shown in FIG. 19.

The VH and VL regions of human monoclonal antibodies specific for rabiesvirus obtained by any of the methods described in the previous examples,can be inserted into an eukaryotic expression vector containing theHATV20 leader sequence and all the coding sequences of the constantregions of human immunoglobulin heavy (for example, IgG1) and lightchains (for example, a kappa light chain) essentially as described (E.Boel et al. (2000), J. Immunol. Methods, 239:153-166). In this example,the following variable region genes optimized for pairing are used:VH-M57, VH-JB (non-modified variable region genes, from Example 2),VH-JA* (the optimized sequence of the VH of antibody JA, from Example5), and only one light chain, VL=M57=JB (from Example 4). The resultingplasmids encoding heavy and light chains are transfected into eukaryoticcells such as the human cell line PER.C6® and in Chinese Hamster Ovary(CHO) to generate stable cell lines secreting antibodies. For this,published methods and methods known to persons skilled in the art areused (E. Boel et al. (2000), J. Immunol. Methods, 239:153-166 and WO00/63403). For the generation of stable PER.C6® cells secretingantibodies, PER.C6® cells are seeded in DMEM plus 10% FCS and in tissueculture dishes (10 cm in diameter) or T80 flasks with approximately2.5×10⁶ cell per dish or flask and kept overnight in an incubator at 37°C. and 10% CO₂. The next day, transfections are preformed in separatedishes at 37° C. using Lipofectamine (Invitrogen Life Technologies)according to standard protocols provided by the manufacturer. Theplasmids encoding the monoclonal antibodies can be mixed in variousratios and used at a concentration of 1-10 μg/ml. As controls, cells aresubjected to the transfection procedure in the absence of plasmids.

After four to five hours, cells are washed twice with DMEM and fed withfresh culture medium. The next day, the culture medium is removed andcells are fed with fresh medium containing 500 μg/ml of the antibioticG418. Cells are fed every two or three days with culture mediumcontaining 500 μg/ml of G418. After about 20 to 22 days after initiationof the experiment, a large number of colonies is visible and from eachtransfection, 300 clones are picked and grown individually in 96-wellplates and further expanded in 24-well, 6-well and T25 flasks. At thisstage, cells are frozen in liquid nitrogen and production levels ofrecombinant immunoglobulin are determined in an ELISA according tostandard procedures (e.g., E. Boel et al. (2000), J. Immunol. Methods,239:153-166 and WO 00/63403). At this stage of the culture procedure,G418 is no longer added to the culture medium.

To establish the presence of anti-rabies antibodies in a mixture, asolid phase anti-rabies ELISA is performed. For the rabies virus ELISA,rabies virus glycoprotein is purified according to standard procedures(Dietzschold et al., in F.-X. Meslin et al. eds., Laboratory techniquesin Rabies, World Health Organization, Geneva, page 175). Plates(PolySorb™, Nunc) are coated with 5 μg/ml of glycoprotein diluted in PBSand 150 μl/well. The plates are then blocked with 5% powdered milk inPBS and washed in PBS containing 0.05% TWEEN®-20 (PBS-TWEEN®) prior tothe addition of supernatant samples. Following incubation at roomtemperature for two hours, the plates are washed with PBS-TWEEN® toremove unbound antibody present in the supernatant samples.Enzyme-conjugated or biotinylated secondary antibodies specific forvarious human heavy chain isotypes are added for one hour at roomtemperature and the plates are subsequently washed with PBS-TWEEN®.Detection of secondary antibody is performed according to standardprocedures (e.g., J. M. Champion et al. (2000), J. Immunol. Methods235:81-90); see also previous examples. Other analysis methods aredescribed in Examples 3, 4 and 12.

Next, it is demonstrated that a clonal cell line accounts for theproduction of each of the binding specificities encoded by the plasmids,i.e., proving that a single cell is able to produce a mixture ofmultiple anti-rabies antibodies. For a limited number of colonies thatsecrete a mixture of all monoclonal antibodies, 30 clones selected fromthe initial panel of approximately 300, clonality is further establishedby subcloning under limiting dilution known to persons skilled in theart. Picked and expanded colonies are seeded in a 96-well plate at aconcentration of 0.3 cells/well in DMEM with 10% FCS and expanded.Colonies of cells are processed as described above and are referred toas subclones. Subclones are screened by PCR on genomic DNA for thepresence or absence of each of the three constructs. Furtherconfirmation of the presence of the constructs is obtained by nucleotidesequence analysis of the PCR products.

For a representative number of subclones, larger volumes are cultured topurify the recombinant human IgG1 fraction from the conditionedsupernatant using Protein A affinity chromatography according tostandard procedures. Purified human Ig from the various subclones issubsequently analyzed by SDS-PAGE, Iso-electric focusing (IEF) accordingto standard protocols (see, also, Examples 3 and 12).

Subclones that are shown to harbor the relevant plasmids are broughtinto culture for an extensive period of time to determine whether thepresence of the plasmids is stable and whether expression of theantibody mixture remains the same, not only in terms of expressionlevels, but, in particular, the ratio between the various antibodiesthat are secreted from the cell. Therefore, the subclone culture ismaintained for at least 25 population doubling times. At every four tosix population doublings, a specific production test is performed usingthe human Ig-specificELISA and larger volumes are cultured to obtain thecell pellet and the supernatant. The cell pellet is used to assess thepresence of the three constructs in the genomic DNA, either via PCR,Southern blot and/or FISH. The supernatant is used to purify therecombinant human Ig fraction as described. Purified human Ig obtainedat the various population doublings is subsequently analyzed asdescribed, i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding inthe inhibition ELISA.

Example 11 Method for Selecting Antigen-Specific Proteinaceous Compoundsusing Mixtures of Encoding DNA

The basis for the mixtures of antibodies present in OLIGOCLONICS® areimmunoglobulin variable regions that encode human monoclonal antibodiesthat have been selected for their specificity, contain variable regiongenes with compatible pairing behavior and are thus compatible with theOLIGOCLONICS® format. For example, antibodies that are encoded bydifferent VH genes and bind to different epitopes but share the same VLgene are suitable for the OLIGOCLONICS® format. Example 7 describes howsuch antibodies are obtained.

In this Example, methods using eukaryotic expression systems to obtainhuman monoclonal antibodies with desired specificities and that sharethe same VL gene are described. Such “repertoires” of human VH genes arePCR-amplified from the B lymphocytes of human individuals and typicallyharbor 10⁸ to 10¹° members. The method is known to persons skilled inthe art and has been described many times in the literature; theamplification of antibody genes is also exemplified for human V-lambdalibraries in Example 4. The source of B lymphocytes may be any lymphoidorgan including blood, bone marrow, tonsil, spleen, lymph node, etc. Theindividual may be pre-selected because it is expected that B lymphocytesproducing the antibodies of interest are enriched in those individualsbecause of, e.g., a prior infection with a micro-organism or because ofa prior immunization, or may be randomly picked. The VH genes may beused unaltered in their coding region or may be altered, particularly inthe CDR3 region to introduce novel specificities. Such VH genes areknown in the art as synthetic or semi-synthetic VH regions. In certainembodiments, primers are used that selectively amplify members of a fewVH gene families such as the large VH3 and VH4 gene families. Primersthat amplify members of more VH gene families may also be used inprocedures known by persons skilled in the art.

Amplified VH regions are cloned into the eukaryotic expression vectorfor human monoclonal antibodies as described in Example 10 andsubsequently introduced into eukaryotic cells such as CHO cells orPER.C6® cells. The expression plasmid shown in Example 10 that harbors aVL gene is used (FIG. 7). There are two alternatives: (1) the VL gene isco-transfected with the VH genes on a separate plasmid or (2) anapproach particularly suitable when only one VL needs to be expressedthe eukaryotic cells are already transfected with a human VL gene thatis stably expressed. The eukaryotic cells are transfected with therepertoire of human VH genes cloned into the eukaryotic expressionvector for human antibodies. High plasmid DNA concentrations are used totransfect the eukaryotic cells in order to introduce multiple copies ofVH genes into a single cell. As a result a single cell will producemultiple antibodies, e.g., between 10 to 1000 different humanantibodies. In the first approach, transfections are transient. In brieffor PER.C6® cells, an 80 cm² tissue culture flask with cells istransfected by incubation for four hours with 140 μl lipofectamine+10 to1000 μg plasmid DNA in serum-free DMEM medium. After four hours, themedium is replaced with DMEM+10% FCS, and the cells are grown overnightat 37° C. Cells are then washed with PBS and the medium is replaced withExcell 525 medium (JRH Bioscience). The cells are seeded at aconcentration that results in the outgrowth of approximately 100transfected cells/well of a 96-well culture plate. After five to sixdays, the cell culture supernatant is harvested and assayed for thepresence of specific antibody by solid phase ELISA. The cells thatcorrespond to the supernatants that score positive in ELISA areharvested and the VH genes are amplified by PCR. Subsequently, theamplified VH genes are cloned into the eukaryotic expression vector forhuman monoclonal antibodies, described in Example 10. Thus, a restrictedrepertoire of human VH genes is obtained. In this example, 100 cellsthat each harbors 100 VH genes yield a maximum of 10⁴ VH genes. Thisrepertoire is transiently transfected into PER.C6® cells that harbor thesame VL gene using low plasmid DNA concentrations (0.1 to 1 μg/ml) suchthat on average a single cell harbors a single VH gene and transfectedcells are plated out under conditions such that only approximately tencells/well will grow out. After five to six days, supernatants arescreened in ELISA for specific antibodies and the cells corresponding topositive supernatants are harvested and used for PCR amplification ofthe VH genes. In this example, the maximum number of VH genes obtainedis approximately ten. Each VH gene is separately transfected intoPER.C6® cells and the VH gene encoding the desired antibody specificityis identified by screening the supernatants of clones in ELISA.

In a second approach, the initial library of 10⁸ to 10¹⁰ VH genes clonedtogether with a single VL gene into the plasmid described in Example 10,is transfected into PER.C6® cells and plated out in T80 cell cultureflasks. After four to six days, the cells are harvested and mixed withred blood cells coated with the antigen of interest and individual cellsare monitored for the secretion of specific antibodies against thecoated antigen by the reverse hemolytic plaque assay, well-known in theart (e.g., F. Dammacco et al. (1984) Clin. Exp. Immunol. 57:743-51). Blymphocytes inducing plaques are visualized under a light microscope andpicked with a micromanipulator. The single transfected PER.C6® cell istransferred to an Eppendorf tube, lysed and subjected to single cell PCRto amplify the VH genes. The advantage of this approach is that only afew rounds of selection are needed to identify the VH gene of interest.

In a third approach, stable transfectants are used. After thetransfection as described above, large collections of clones are grownessentially as described in Example 10, with the exception that clonesare not plated out under limiting dilution conditions. Instead, thecells after transfection are plated in microtiter plates such that aftergrowth in the selective medium multiple clones per well arise (e.g., 100cell clones per well as indicated in FIG. 7). Each clone expressesmultiple species of heavy chains, and each well contains multipleclones. The supernatant of these cultures are tested for antigen bindingand the VH-genes are further enriched in cycles of PCR, cloning,transfection and screening, as described above.

The expression of multiple antibodies by a single transfected eukaryoticcell is improved in all of these approaches by introducinganti-repressor DNA elements in the plasmid constructs for the expressionof human monoclonal antibodies. Anti-repressor elements confer highlevel and stable expression of proteins in mammalian cells in a copynumber-dependent fashion (Kwaks et al. (2003), Nat. Biotechnol.21:553-558). The DNA fragments responsible for this effect are amplifiedfrom the clones described in this citation and introduced upstream ofthe heavy chain expression cassette. The human anti-repressor elementnr. 40 (SEQ ID NO:117) is amplified from the pSDH vector containing theelement (described in Kwaks et al.), using flanking oligonucleotidesthat also incorporate restriction sites suitable for cloning(5′-GTCCCTAGGAATTCGATCAAGAAA GCACTCCGGG-3′ (SEQ ID NO:144) and thereverse complement of 5′-CCTCATGATGTACATTAGAT CGAATTCGTAATACG-3′ (SEQ IDNO:145)). In this example, EcoRI (GAATTC (SEQ ID NO:146)) which is notpresent in this segment, is appended at both ends of the segment in aPCR reaction, and the fragment digested with EcoRI and cloned into anEcoRI-digested acceptor plasmid. In this example, the latter is achimeric plasmid of VHExpress and VLExpress, which is a composition madeby cloning the full VHExpress plasmid (FIG. 15), cut with Kpnl andEcoRI, and inserting the VK expression cassette that was digested withthe same enzymes (described in Persic et al. (1997) 187:9-18). Theresulting plasmid, pABExpress40 contains both heavy and light chaincassettes with their respective transcriptional orientation in oppositedirections, and the anti-repressor element positioned in the middle ofthe two transcription units. A schematic map of the plasmid is shown inFIG. 22. This plasmid, pABExpress40 is used first in the cloning of theone chosen VL gene (using ApaLI and PacI cloning sites), resulting inpABExpress40-VL. This plasmid is used to receive the VH repertoiredescribed above (as BssHII-BstEII fragment) (all of these four sites areunique in pABExpress40 and pABExpress40-VL). The cloning of therepertoire is carried out as described for the lambda repertoire inExample 4, using in the PCR of IgM-primed cDNA a set of nineoligonucleotides labeled “VH-back” and the mix of four “VH-forward”oligonucleotides described in Table 1 of H. J. de Haard et al. (1999),J. Biol. Chem. 274:18218-18230. The material is re-amplified usingvariants of the nine oligonucleotides appended with 5′-TATC CGC GCG CACTCC-3′ (SEQ ID NO:147) and with the same VH forward mix, the productdigested with BssHII and BstEII and cloned into pABExpress40-VL. Thelibrary is subsequently used as described in the previous examples toisolate panels of antigen-binding clones. Similarly the vector is usedto construct the expression plasmid for given sets of antibodies, suchas the ones described in Example 10, further confirming that theflanking variable region genes by anti-repressor elements facilitatesthe efficient and stable production of multiple antibodies by a singlecell.

Example 12 Recovery and Analysis of Antibody Mixtures using ELISAIncluding the use of Anti-Idiotype and Peptide Mimotopes

Antibody mixtures containing Fc regions are recovered as indicated inExample 3 using Protein A affinity chromatography. Antibody fragmentswith Histidine tags are isolated using IMAC as described in Example 2.

The resulting protein mixtures are analyzed as follows. Considered wasthe case of an antibody mixture composed of different binding sitesdirected to the same target antigen, with all antibodies being the sameisotype, carrying the same light chain, and the mixture containing bothmonovalent bispecific and bivalent monospecific IgG-type antibodies. Thefollowing methods are available for analyzing the mixture. The heavychain variable region genes will yield different amino acid compositionsand allow the following non-antigen-dependent analysis: (1) Isoelectricfocusing gel analysis: this analysis relies on a different pI value forthe various forms of the antibodies. In a mixture of two IgGs and onebispecific, these three molecules will each display a unique isoelectricpoint. Proteins with a different pI are separated via electrophoresis ina pH gradient. The method is semi-quantitative. If two proteins of thecomplex have only a minimal difference in their pI value, it will bedifficult to separate them using this test, and the other tests citedare used. (2) Mass-Spectrometry analysis: this analysis relies on thedifferential composition of the VH region, which, after digestion withproteolytic enzymes, yields a unique spectrum of peptides in MassSpecanalysis. This method is predominantly qualitative. (3) Binding analysisbased on anti-idiotype antibodies or peptide mimics: this analysisrequires the availability of reagents that specifically recognize oneantibody binding site in the presence of the other binding sites in themixture. Suitable for this analysis are anti-idiotype antibodies whichuniquely recognize the idiotype of the antibody. In this example wherethe antibodies share a common light chain, the unique features of theidiotype are formed mainly by the heavy chain determinants Anti-idiotypeantibodies are selected using the individual monoclonal antibodies asantigen in a selection of a large phage displayed antibody library usingmethods known to those in the art. Typically used are a non-immuneantibody library (H. J. de Haard et al. (1999), J. Biol. Chem.274:18218-18230), which yields Fab fragments, and a semi-synthetic phageantibody library (de Kruif et al. (1995) J. Mol. Biol. 248:97).Anti-idiotype antibodies are selected on immobilized M57 and JBantibodies from the cited non-immune antibody library. Using ELISAscreening of the selected phage antibodies on these two monoclonalantibodies used for the selection, anti-idiotype antibodies thatuniquely recognize one of the two binding sites are identified. Therespective Fab and scFv reagents selected from these library, areexpressed as antibody fragments and purified using standard methods, forexample, described in these citations and in Antibody Engineering(2001), Eds. Konterman and Dubel, Springer Lab Manual, and described inExample 2 for the scFv antibodies. The fragments are used in ELISA todetermine which idiotype is present in the mixture, which is carried outin a quantitative assay. The anti-idiotype antibodies specific for thebinding sites of M57 and JB are also used in virus competitionexperiments with the OLIGOCLONICS® preparation made in Example 10, todelineate the contribution of an individual binding site to thebiological activity of the antibody mixture. Alternatively, themonoclonal antibodies are used to derive idiotype-associated peptides,linear or conformational peptides derived from the sequence of theantigen and still reactive with the antibody, for example, via PepScananalysis, as was demonstrated for the rabies virus neutralizing antibodyMAb 6-15C4 (van der Heijden et al. (1993), J. Gen. Virol. 74:1539-45).An alternative is to isolate peptide mimotopes, with sequences unrelatedto the original antigen yet specifically binding to the variable regionsof the antibody. Provided the reaction is specific for a given antibodyin the context of the other antibodies in the mixture, such peptides arealso suitable for a specific analysis of the antibody mixture. Peptideswith such unique reactivity to a given antibody are selected from phagedisplay peptide libraries using methods essentially similar to those forphage antibody libraries. The binding test with the anti-idiotypeantibodies and peptide-mimotopes is qualitatively or quantitatively, anda large series of binding tests are feasible, including ELISA, RIA, Flowcytometric analysis, BIAcore, etc.

Also disclosed is the analysis of an OLIGOCLONICS® mixture comprisingmultiple antibodies, in which each of the original antibodies binds to adifferent antigen. This resembles the situation in which the antibodiesrecognize the same antigen or target, and anti-idiotype reagents orpeptide mimics are available. The analysis of multiple specificities ina mixture is carried out as follows, keeping in mind that antigen issynonymous for anti-idiotype. The reactivity to individual antigens istested in ELISA on all antigens separately, with standardized assaysusing the monoclonal antibodies and quantitative IgG ELISA test. Antigenis coated directly or indirectly, the plates incubated with the antibodymixture, and bound antibody detected with an anti-IgG reagent. Thisleads to a “specific” activity of the preparation, that is a reactivityin relative units of activity per antibody quantity. The amount ofbispecific antibody in the mixture is determined using a sandwich assaywith one antigen coated and a second antigen, for example labeled withHRP, Alkaline Phosphatase or biotin, or detectable using anotherantibody specific for this antigen, provided to the plate after theantibody mixture was incubated with the first antigen.

If the antibodies present in the OLIGOCLONICS® mixture are bindingdifferent targets or different epitopes on the same target such thatthey are non-competitive, this feature can be used in an inhibitionELISA to determine the presence of the different antibodies in themixtures produced by the transfected clonal cell lines. Consider anOLIGOCLONICS® made according to the methods of the previous examplesusing the antibodies specific for the Rabies glycoprotein isolated inExample 7 (which are non-competitive). For the inhibition ELISA, thesame procedures as described for the standard anti-rabies ELISA asdescribed above is used with some modifications. The OLIGOCLONICS®mixture produced by a clonal cell line is characterized as follows.Before addition to the wells coated with rabies glycoprotein, thesupernatants of the transfected clonal cell line is diluted with anequal volume of a biotinylated rabies monoclonal antibody used to makethe mixture. The biotinylated rabies monoclonal antibody is added invarious concentrations, ranging from 0.1 to 10 μg/ml. Binding of thebiotinylated monoclonal antibody to the coated rabies glycoprotein isinhibited when the same non-biotinylated antibody is present in themixture produced by the clonal cell line. The binding of thebiotinylated antibody is visualized with streptavidin, conjugated to anenzyme. As a control for binding and degree of inhibition, variousconcentrations of the biotinylated monoclonal antibodies diluted with anequal volume of culture medium without the mixture of antibodies orusing the non-biotinylated antibody are used in the inhibition ELISA.This method is also suitable to screen the mixture of antibodies at avery early stage after transfection (as in Examples 10 and 11); thus,for each supernatant containing mixtures of antibodies, the presence ofindividual antibody specificities can be determined.

Example 13 Expression of Three Fab Fragments in the same Eukaryotic Cell

For making a mix of these three antibodies, the expression experimentdescribed in Example 10 is repeated using the following antibody genes,of the M57, JB and PO1 antibody (the latter is formed by the VH-PO1 andVL-PO1 genes of Example 9). Anti-idiotype reagents are separatelyselected on M57 and JB whole antibodies using a non-immune antibodylibrary (see, also Example 12). This yields anti-idiotype antibodiesthat react with either M57 or JB; these antibodies are also tested onthe PO1 to confirm specificity for either M57 or JB idiotypes.Similarly, the PO1 antibody is used in similar selections to obtain ananti-Id reagent for the PO1 binding site. Next, the heavy chains ofthese three antibodies, M57, JB and PO1, are cloned as VHCH1 fragmentsinto VHExpress while deleting the gamma-1 gene (thus encoding an Fdchain only), yielding pEU-VH-M57, pEU-VH-JB and pEU-VH-PO1. The lightchains VL-M57=JB-CL and VL-PO1-CL are cloned into VKexpress (Persic etal. (1997) 187:9-18), while deleting the CK gene from the cassette.First the light chain plasmids are introduced into PER.C6® cells and aclone is selected that stably produces over 2 micrograms/ml of bothlight chains (using methods described in Example 10). This cell line,designated PL2-2, is subsequently transfected with the three heavy chaincontaining plasmids, and a large collection of cell lines is obtainedthat produce a variety of levels of antibody Land Fd chains. The bestcandidate mixtures are purified on protein G affinity chromatography andtested for binding and composition as described in the previousexamples, and also using the anti-Id reagents as described in Example12. The experiments provide confirmation that multiple Fab fragments,with appropriately paired variable region genes, are expressed as highlyfunctional mixtures.

Example 14 Cloning and Expression of Three Antibodies Directed toDifferent Antigens as an OLIGOCLONICS® Mixture

Using the methods of the previous examples, antibodies with the samelight chain are isolated against three different antigen, TNF-alpha,Interleukin-lbeta (IL- lbeta) and Interleukin-6 (IL-6), using asemi-synthetic library scFv library from Example 7 and described in (deKruifet al. (1995) J. Mol. Biol. 248:97). In the selection, biotinylatedrecombinant cytokines (purchased from R&D Systems), are used, atdecreasing concentrations during selection (250 nM, 100 nM and 50 nM).From the panels of antibodies generated against each of the targetsafter three rounds of selection, those scFv antibodies that neutralizethe activity of the cytokine are identified. For this, the antibodyfragments are recloned into pSCFV and purified using IMAC as in Example2. Biological assays used are well known to those skilled in the art andinclude a L929 neutralization assay for TNF-alpha. Neutralizing clonesare identified against TNF-alpha, IL-lbeta or IL-6. The potency ofneutralization can be improved by further affinity maturationtechniques. For example, the CDR1 and CDR2 of the VH can be mutagenizedand variants selected using phage display and tested for improvedneutralization activity. These three antibodies have an identical lightchain and have heavy chain variable regions that are distinct from oneanother, with most changes located in the CDR3.

The antibody variable regions are cloned into the eukaryotic expressiondescribed in Example 10, and essentially following the same procedure,CHO-cell lines identified that express mixture of the one light chainand three heavy chains The analysis of the mixtures is carried out usingELISA to demonstrate binding to three antigens in a subset of the celllines identified. A clone stably producing all three antibodies in anapproximate ratio of heavy chains of 2:1:1 is identified using thetechniques described in Examples 10 and 12. The cell lines are expandedand the mixture purified on Protein A and extensively tested todetermine its composition. Using ELISA tests in various formats, withindirectly coated biotinylated antigen, with directly coated antigen 1,adding sample, followed by biotinylated antigen 2 and detection withStrep-HRP, and using samples of the mixture that have been depleted onTNF, IL-lbeta or IL-6-coated beads, is it shown that the mixturecontains three monospecific antibodies and three bispecific antibodies.The exact ratio between these six components is established by usingquantitative ELISA tests and by IEF analysis of the mixture, as shown inExample 12. The neutralization efficacy of the mixture for theindividual cytokines was confirmed with the assays as tested before. Theneutralization of these cytokines in more complex systems, for example,using mixed cell populations, may establish a synergistic effect of theneutralization of these components by the OLIGOCLONICS® mixture.

Example 15 In Vitro Pairing of Antibody Chains Produced in DifferentCells to Form Defined Antibody Mixtures

Alternatively, to the expression in one host cell, antibody mixture canalso be assembled ex vivo. The chains can be expressed separately andcombined with a set of potential partner variable regions for pairingand assembly of the molecule.

In this prophetic example, a mixture of Fab fragments withpairing-compatible variable regions will be made as follows. The heavychain variable regions of M57, JB and PO1 (Example 9) will first becloned separately into an appropriate pET expression plasmid, such thatthis will mediate the expression of the Fd chain tagged with sixHistidines inside the E. coli, as inclusion bodies. A suitable vectorcan be found in Novagen's pET Table, such as pET21d+(see, alsowww.novagen.com/Includes/wrapper.asp?href=/SharedImages/Novagen/pETtablehtm&section=TechResources&subsectjon=TechLit&strsubsection=techresources).The cloning will then be carried out by PCR of the VHCH1-containingtemplates (from Example 9) using oligonucleotides to provide appropriatecloning sites and also the C-terminal Histidine tag. These threeplasmids will be introduced into separate E. coli host cells. Theexpression of the Fd fragments can then be induced and the proteindemonstrated to be present in inclusion bodies. The two light chainvariable regions, VL-M57=JB and YL-PO1 can also be suitably cloned intoa suitable pET vector (although, alternatively, they could be obtainedby secretion from a secretion vector like pFab-sol-pbr). Afterexpression of the separate light chains, they should also be retrievablefrom the intracellular fraction. To assemble the mixture of threefunctional Fab fragments, the following procedure can then be followed.First the approximate and relative quantities of the individual L or Fdchains is estimated by gel-electrophoresis and Western blot. Then five50-ml cultures of E. coli carrying one of five antibody variable regionsare grown and induced as described in the pET manual from Novagen. Afterinduction and growth, the pelleted cells of each of the chains can beresuspended in 8 ml 8 M urea (in PBS). After sonication, the fivesuspensions would be mixed in a ratio of approximately 1:1:1:4:2 forVH-M57, VH-JB, VH-PO1, VL-M57=JB, VL-PO1 (thus with a two-fold excess oflight chain over heavy chain, and more of the twice needed VL). Afterthis mixing of the denatured heavy and light chain variable regions, themix will be rotated head over head for two hours. Insoluble material maythen be removed by centrifugation for 30 minutes at 13,000×g. Thesupernatant is dialyzed against PBS with four buffer changes, andinsoluble protein further removed by centrifugation. The flow throughfraction, obtained by filtration through a 0.2 μm membrane, shouldcontain the refolded antibody mixture with pairing-optimized chains Themixture may be further concentrated and purified, first using IMAC,which should retrieve all heavy chains and their paired light chains,followed by semi-preparative gel-filtration on a Superdex 75HR column(Pharmacia). The yield may be determined by measuring the opticaldensity at 280 nm (using a molar extinction coefficient of 13 for Fabs).The mixture may be further characterized by analyzing the binding to theRabies antigen. Since all functional Fabs should bind this antigen, astraightforward capture assay with antigen may be performed to determinethe level of functional binding sites. There are many alternativeprotocols to this procedure, including the use of other extractionmethods, other denaturation reagents, renaturation conditions andbuffers, etc. Alternatively, to this procedure, both chains may also besecreted, and re-assembled using the conditions described by Figini etal. (1994) J. Mol Biol. 239:68-78.

Example 16 Screening Antibody Mixtures Targeting Murine VascularEndothelial Growth Factor

The antibodies used in this example are described in WO 03102157A2(inventors Fuh and Sidhu). The antibodies were derived by in vitroselection of a display library in which only the heavy chain wasdiversified. The repertoire with a fixed light chain and variable heavychain was selected on murine vascular endothelial growth factor (mVEGF)and a large panel of antibodies binding mVEGF identified (Sidhu et al.,J. Mol. Biol. 2004, 338:299-310). The source of the antibody heavy andlight chain variable genes used in the repertoire was the humanizedantibody 4D5. Antibody 4D5 is a humanized antibody specific for acancer-associated antigen known as Her-2 (erbB2). The antibody includesvariable domains having consensus framework regions; a few positionswere reverted to mouse sequence during the process of increasingaffinity of the humanized antibody. The sequence and crystal structureof humanized antibody 4D5 have been described in U.S. Pat. No.6,054,297, Carter et al., PNAS 54:4285 (1992); the variable regionsequences of the heavy and light chains are also given in FIGS. 14A and14B and SEQ ID NO:23 of WO 03102157A2; finally the crystal structure of4D5 is shown in J. Mol. Biol. 229:969 (1993) and online atwww.ncbi.nih.gov/structure, structure 1FVE.

An OLIGOCLONICS® mixture consisting of four different mVEGF-bindingantibody binding sites is obtained as follows. Antibodies with clonenumbers 4, 69, 73 and 74 as in Table 6, page 306 of Sidhu et al., J.Mol. Biol. 2004, 338:299-310, were selected on the basis of mVEGFbinding as scFv on phage and as Fab protein (same Table 6). Theantibodies share an identical light chain (of the Herceptin antibody,4D5; as described in WO 03102157A2), but have differences in their heavychain amino acid sequence as depicted in Table 6 of this paper.

The h4D5 antibody is a humanized antibody that specifically recognizes acancer-associated antigen known as HER-2 (ErbB2) developed as describedpreviously. The h4d5 VL gene is obtained by polymerase chain reactionusing the humAb4D5 version 8 (“humAMD5-8”; Carter et al. (1992) PNAS89:4285-4289) sequence and primers engineered to give rise to a 5′ ApaLIsite and a 3′ PacI site in the PCR product. The PCR product was cleavedwith ApaLI and PacI and ligated into the pABExpress vector (the vectordescribed in Example 11 and in FIG. 23 but without the STAR40 sequencecloned into the EcoRI site). This yields plasmid pAb-4D5-VL, whichencodes the expression of a functional 4D5 light chain (with humanCkappa constant region), and contains a polylinker region suitable forcloning VH regions. The VH regions from clones 4, 69, 73 and 74 are thencloned into this vector, using BssHII and BstEII restriction sites, andfollowing the cloning route described in the previous examples (byproviding the nucleotides encoding these restriction sites into the PCRprimers in such manner that the cloning will yield an in-frame insertionencoding a fully functional antibody variable domain) This yieldsplasmids pAb-IgG-04, pAb-IgG-69, pAb-IgG-73 and pAb-IgG-74.

These plasmids encoding heavy and light chains are transfected into thehuman cell line PER.C6® to generate stable cell lines secreting multipleof the mVEGF-binding antibodies. For this, published methods and methodsknown to persons skilled in the art are used (E. Boel et al. (2000). J.Immunol. Methods, 239:153-166 and WO 00/63403). For the generation ofstable PER.C6® cells secreting antibodies, PER.C6® cells are seeded inDMEM plus 10% FCS and in tissue culture dishes (10 cm in diameter) orT80 flasks with approximately 2.5×10⁶ cell per dish or flask and keptovernight in an incubator at 37° C. and 10% CO₂. The next day,transfections are preformed in separate dishes at 37° C. usingLipofectamine (Invitrogen Life Technologies) according to standardprotocols provided by the manufacturer. The plasmids pAb-IgG-04,pAb-IgG-69, pAb-IgG-73 and pAb-IgG-74 are mixed in a 1:1:1:1 ratios andused at a concentration of 2.5 μg/ml each. As controls, cells aresubjected to the transfection procedure in the absence of plasmids, orwith just a single plasmid. After four to five hours, cells are washedtwice with DMEM and fed with fresh culture medium. The next day, theculture medium is removed and cells are fed with fresh medium containing500 μg/ml of the antibiotic G418. Cells are fed every two to three dayswith culture medium containing 500 μg/ml of G418. After about 20 to 22days after initiation of the experiment, a large number of colonies isvisible and from each transfection, 400 clones are picked and grownindividually in 96-well plates and further expanded in 24-well, 6-welland T25 flasks. At this stage, cells are frozen in liquid nitrogen andproduction levels of recombinant immunoglobulin are determined in anELISA according to standard procedures (e.g., E. Boel et al. (2000), J.Immunol. Methods, 239:153-166 and WO 00/63403). At this stage of theculture procedure, G418 is no longer added to the culture medium.

To establish the presence of at least one functional anti-mVEGF antibodyin a clone's culture supernatant, a solid phase ELISA is performed.Plates (PolySorb™, Nunc) are coated with 2.5 μg/ml of mVEGF (R&DSystems, recombinant Mouse VEGF120 and VEG164, both carrier free)diluted in PBS and 100 μl/well overnight at 4° C. The plates are thenblocked with 2% BSA in PBS for two hours and washed in PBS containing0.05% TWEEN®-20 (PBS-TWEEN®) prior to the addition of cell supernatantsamples containing antibodies. Following incubation at room temperaturefor two hours, the plates are washed with PBS-TWEEN® to remove unboundantibody present in the supernatant samples. Horseradishperoxidase-conjugated anti-human IgG is then added in PBS for one hourat room temperature and the plates are subsequently washed withPBS-TWEEN® (2×) and PBS (2×). Detection of secondary antibody isperformed according to standard procedures and the absorbance determinedspectrophotometrically (see, also previous examples). It is found thatof the 400 clones screened, a substantial fraction produces a minimalIgG quantity.

Since only a limited number of colonies secrete a mixture of the fourmVEGF antibodies, 50 clones selected from the initial panel ofapproximately 400, that are strongly reactive in the IgG-ELISA,clonality is further established by subcloning under limiting dilution.Picked and expanded colonies are seeded in a 96-well plate at aconcentration of 0.3 cells/well in DMEM with 10% FCS and expanded.Colonies of cells are processed as described above and are referred toas subclones. While the initial transfection experiment used a ratio ofDNA for the four plasmids pAb-IgG-04, pAb-IgG-69, pAb-IgG-73 andpAb-IgG-74 of 1:1:1:1, the cell subclones still display a variety in theexpression levels for each of the antibodies. This is due to theirindependent expression regulation and their random integration into thegenome. Further, since the same selection marker is used on allplasmids, the subclones express at the most four antibody binding sites,but not necessarily all four of them. The precise number depends on thetransfection experiment; approximately 20-30% of the Ig-reactive clonesexpress multiple antibody heavy chains, and of these, approximately 20%express more than two antibody heavy chains. The methods to increasethese frequencies have been described earlier herein.

Screening to find the most optimal mixture of these four mVEGF-bindingantibodies, as OLIGOCLONICS® mixture with bivalent and bispecificcomponents, is done as follows. Optimal mixture here means with regardsto which of the four antibody binding sites are optimally present in themixture, and at which ratio they should be present. For the 50 subclonesas well as for one IgG-reactive clone from the control transfectantsmade with just one antibody encoding plasmid, larger volumes arecultured to purify the recombinant human IgG1 fraction from theconditioned supernatant. This is done using Protein A affinity columnchromatography according to standard procedures (Ed Harlow and DavidLane, Using Antibodies, A Laboratory Manual, 1999, ISBN: 0879695447).These mixtures and the monoclonal antibody controls are tested for theirneutralization activity on mVEGF in a ³H-thymidine incorporation assayusing human umbilical vein endothelial cells (Conn et al., 1990, Proc.Natl. Acad. Sci. U.S.A. 87:1323-1327). The inhibitory activity of eachof the mixtures is compared to the inhibitory capacity of the fourindividual monoclonal antibodies. Mixtures that display a higherinhibitory activity on a molar basis compared to the activity of themonoclonal antibody controls putatively contain multiple antibodies thatin combination mediate a synergic effect on the activity of VEGF. Next,assays that indicate the binding to mVEGF, the affinity of theinteraction of the mix, the competition in binding with receptor (Flt-1and KDR-1), are used. A binding assay is described above (solid phaseELISA). Assays to determine the relative affinity of the mixes aredescribed in Sidhu et al., J. Mol. Biol. 2004, 338:299-310, page 308(affinity measurements by competitive ELISA), with Fab andphage-displayed antibodies replaced with the mixtures of antibodies orthe monoclonal antibodies as controls. An increase in relative affinityindicates a strong synergistic activity between the antibodies in themixture, as described in Marks, Movelent Disorders, vol. 19, suppl. 8,2004, p. S101-S108, for antibody mixtures binding to nonoverlappingepitopes of botulinum neurotoxins. Other assays to demonstrate theactivity of the mixture of the antibodies on VEGF either in vivo or invitro, are well established in the field and are, for example, describedin WO 03102157A2, EP 0666868B1 and WO0044777A1.

Since VEGF displays activities in many processes, including mitogenesis,angiogenesis, endothelial cell survival, induction of metalloproteinasesand growth factors, regulation of permeability/flow, recruitment ofendothelial progenitor cells etc, any other single assays orcombinations of assays can be used to determine the effect of theantibody mixtures on the activity of VEGF. The antibody mixtures can bescreened in any of these assays, or combinations of assays, to findthose compositions that have an effect in a defined set of assays, orhave an effect in one but not in another assay. Further or instead ofthe in vitro assays, in vivo assays can be used to measure the overalleffect of the antibody mixture on the pharmacokinetics of the antigen,and demonstrate improved clearance as mechanism of the synergic activityof the multiple antibodies in the OLIGOCLONICS® mixture.

Mixtures are further characterized biochemically to find whichantibodies are present and in which ratio, as described in Example 12.

Example 17 Pairing-Compatible Antibodies for Producing a Mixture ofHER2/ErbB2-Targeting Molecules

Trastuzumab (Herceptin, or h4D5, or hu4D5, see Example 16) andpertuzumab (Omnitarg, humanized 2C4) are both recombinant monoclonalantibodies that target different extracellular regions of the HER-2tyrosine kinase receptor. Recently, it was shown that these antibodiessynergistically inhibit the survival of breast cancer cells in vitro(Nahta et al., Cancer Research 64:2343-2346, 2004). Herceptin is activeagainst HER-2 overexpressing metastatic breast cancers, leading to itsapproval in 1998 by the US FDA. In contrast to Herceptin, pertuzumabsterically blocks HER-2 dimerization with other HER receptors and blocksligand-activated signaling from HER-2/EGFR and HER-2/HER-3 heterodimers.On the other hand, trastuzumab blocks ErbB2 shedding while pertuzumabdoes not. Mixtures of antibodies directed to the same target antigen butthat display different or non-overlapping mechanisms of action will bevery valuable in the therapeutic arsenal, and production of suchmultiple antibodies in a commercial manner will become very important.In this example, described is how pairing-compatible versions of thesetwo antibodies are isolated, and used to build an OLIGOCLONICS® with anexpected increase in potency and efficacy in tumor cell killing comparedto the original monoclonal antibodies.

Anti-HER2 antibodies 4D5 and 2C4 are described in WO 0100245A2 and inFendly et al., Cancer Research 50:1550-1558 (1990). The molecularstructure and sequence of the humanized version of antibody 2C4 isdescribed in Vajdos et al., J. Mol. Biol. 2002, 320, 415-428, in PDBdatabase reference 1L71, and in WO 0100245A2 (version 574 in Table 2 onpage 54, or rhuMAb2C4 in continuation of this document). For simplicityhere “2C4” is used to indicate the humanized version 574 of the murine2C4 antibody. Its structure, in complex with the first three domains ofErbB2, was recently published (Franklin et al., Cancer Cell, 5, 2004,317-328. The structure and sequence of h4D5 or Herceptin was describedby Cho et al., Nature 2003, 421, 756-760, and is deposited as 1N8Z inthe PDB database. Outside of the complementarity-determining regions(CDRs), pertuzumab is identical in sequence to trastuzumab (Carter etal., Proc. Natl. Acad. Sci. U.S.A. 89, 4285-4289, 1992); consequently,the local structure of the pertuzumab Fab in the ErbB2-pertuzumabcomplex is expected to be largely the same as that of the trastuzumabFab. To build a pairing-compatible single light chain that will restorea functional binding site when paired with the h4D5 VH but also whenpaired with the 2C4 VH, the following route is followed.

Designing pairing-compatible light chains: The amino acid differencesbetween the light chains of hu4D5v8 (the humanization variant describedby Kelly et al., 1992, supra, indicated by hu4D5 or h4D5 in the nextsection) and 2C4 have been mapped to be 11 residues as highlighted inFIG. 23. In the CDR regions of the light chains, there are fourdifferences in CDR1, three in CDR2 and four in CDR3. In most straightforward to follow in the absence of structural data on the antibodiesand their interaction with antigen, is to build a library of light chainthat have been diversified at these positions, and screen or select forvariant light chain that maintain antigen-binding behavior when pairedwith the heavy chains of both antibodies, h4D5 and 2C4. Thediversification can be chosen to contain all possible 20 amino acids ora subset thereof, for example, all residues but cysteine (which is notnormally occurring at these 11 positions), or a selected set of aminoacids that frequently occurs in antibodies at these positions. Thedesign of a light chain repertoire based on all 11 amino aciddifferences between h4D5 and 2C4 is given in FIG. 23, in line HYB1.

A second approach to build a pairing-compatible variable hybrid lightchain region for two antibodies, is to further employ structuralinformation on the interaction of the antibodies with their respectiveantigen or antigens. In the example of h4D5 and 2C5, a wealth ofstructure-function information is available to guide the design of ahybrid light chain library. In this design, HYB2 in FIG. 23, all thelight chains in the designed repertoire retain all of the commonresidues between the two original light chains of hu4D5 and 2C4, and aselection of residues at the positions where the original two lightchains differ in composition, in which the selection is based onstructural information on the antibody-antigen interaction. While someof the design may be based on this information, it is also noted thatpoint mutations of h4D5 have been shown to dramatically effect thebiological behavior of the antibody. The antiproliferative activities ofthe humanized variants of 4D5, which differ only in maximally sevenamino acid residues, were found not to be strongly correlated withantigen affinity (Kelley et al., 1992, supra). Thus, it will be requiredto sample multiple versions of pairing-compatible light chains, and testthe biological activity of the combinations after the antigen-selectionand binding characterization to ensure maintenance of the biologicalactivity.

The following HYB2 library design was made, based on the followingobservations:

CDR1. The sequence plasticity of the antigen-binding site of Herceptinwas analyzed in a study by Gerstner et al. (J. Mol. Biol. 2002,321:851-862). From these studies it appears that for trastuzumabresidues N30 may be readily replaced by Serine (Table 1, Class 1mutation VL30, of Gerstner et al., supra). Serine is the residue used atthis position by 2C4. Thus, the pairing-compatible hybrid light chain isdesigned to contain Ser at position 30. The rest of the CDR1 is takenfrom the Herceptin light chain, as this region appears to be irrelevantfor antigen binding in 2C4 (Franklin et al., supra).

CDR2. By alanine-scanning and homolog-scanning of the Fab2C4 antibody itwas revealed that most of the side-chains that contribute to antigenbinding are located in the heavy chain (Vajdos et al., supra). This wasrecently confirmed by the crystal structure of the antibody in complexwith antigen: the light chain of pertuzumab Fab makes only a fewcontacts with ErbB2, mostly via CDR L2 (possibly via residue 55) andsome via L3 (Franklin et al., supra). Some of 2C4′s residues in thisregion may be converted to h4D5′s residues without loss of affinity, assuggested by experiments with humanized versions of 2C4 described in WO0100245A2 (page 54), in particular, what may be possible is to chooseh4D5′s VL's residues at positions 54 and 56. The Phe at position 53 inHerceptin appears to be relatively conserved, with some presence of Trp,while the other positions in this CDR region were not tested. Since someof these CDR2-based residues may also be important for positioningneighboring heavy-chain-based residues for antigen binding, in thehybrid light chain design, the three residues which are differentbetween h4D5 and 2C4 are diversified fully, such that the selectionprocess can identify which of the 8000 combinations will yield apairing-compatible light chain

CDR3. Tyrosine 91 of 2C4 is said to be important for antigen binding(Franklin et al., supra) but its substitution with phenylalanine (F) isacceptable (Vajdos et al., supra). Herceptin at this position in thelight chain besides its original residue histidine tolerates severalother aromatic side chains including Phe, Tyr and Trp (Table 1, page 854in Gerstner et al., supra). Thus, the hybrid light chain is designed tocontain Phe at position 91 (FIG. 23). For 2C4 antigen binding of theother residues of the H3 loop is relatively resistant to mutagenesis asin Gerstner et al., with the exception of the Pro at position 95. Butthis residue is shared between the Herceptin and 2C4 antibody lightchains. In the interaction of Herceptin with antigen there are morelikely interactions of the CDR3 regions with antigen, thus in the hybridlight chain, all but residue 91 is taken from Herceptin-VL (FIG. 23).

In the final HYB2 design, amino acids are taken for 6 out of 11positions from the h4D5 VL, 1 out of 11 from the 2C4 VL (pos. 30), oneis a residue not found in either VL (pos. 91) and the three are to berandomized (in CDR2).

HYB1 Library Construction and Selection of Pairing-Compatible VLs

The two libraries of light chains are constructed as follows. In theHYB1-designed VL library, 11 residues are randomized, implying that thetotal theoretical amino acid diversity (20exp11) is much larger than canbe readily screened. To sample the diversity in this library, a powerfulselection method is, therefore, used. The heavy chains (VH) of h4D5 and2C4 are cloned into the SfiI-BstEll cloning sites from pCES1 (de Haardet al., 1999, J. Biol. Chem. 274, 18218-30) using PCR andoligonucleotides binding to the 5′ and 3′ end of the nucleotidesequences of the VH genes and introducing SfiI and BstEII sites atappropriate sites for in-frame cloning (as described for antibody VHgenes in de Haard et al., supra; the BstEII site is already present inthe JH region of both h4D5-VH and 2C4-VH). The template for the PCR ofthe VH of h4D5 is plasmid pAK19 carrying the humanized 4D5 variantnumber 8, hu4D5-8, described in Kelly et al., 1992, Biochemistry31:5435-5441, Table 1. The nucleotide sequence of this clone isessentially described in Carter et al. 1992, P.N.A.S., 89:4285-4289, inFIG. 1, as huMAb4D5-5, with two alterations (V102Y in CDR3 of the VH,and E55Y in CDR2 of VL, as described in Kelly et al., 1992, supra). TheVH sequence can also be extracted as SfiI-BstEII fragment from SEQ IDNO:119 as described below. The template for the PCR reaction of the VHof 2C4 is plasmid pC2C4, described on page 425 of Vajdos et al., supra.The VH sequence can also be extracted from the Ncol-BstEII insertioninside the larger BssHII-Notl-fragment from SEQ ID NO:119. The cloningof the PCR products into pCES1 is carried out as described for humanantibody heavy chain VH pools and using standard cloning procedures.pCES1 is a phagemid vector that is suitable for the expression of Fabfragments in E. coli and for the display of Fab fragments on the surfaceof filamentous phage (de Haard et al., 1999, supra). Two plasmids withcorrect insert are identified by sequencing the insertion and junctionregion and the resulting plasmids named pCES-VH-h4D5 and pCES-VH-2C4.These are the acceptor plasmids for the light chain repertoire, HYB1.The VLCL coding region of hu4D5v8 is amplified using specificoligonucleotides priming in its 5′ and 3′ region and introducing ApaLIand Asci restriction sites as described in de Haard et al., supra, forhuman VLCL chains. As template pAK19 carrying the humanized 4D5 variantnumber 8 (hu4D5-8, described in Kelly et al., 1992, Biochemistry31:5435-5441, Table 1) is used. The PCR product is cloned as ApaLI-AscIfragment in pCES-WI-h4D5, to yield pCES-Fab-h4D5. This encodes afunctional h4D5 Fab fragment. HYB1 is produced using described methodswith “stop” template versions of this plasmid. The stop template versionis made by replacing one codon in each of the CDR1, CDR2 and CDR3 of thehu4D5-v8-VL with TAA stop codons. Methods to diversify the VL-templatehave been extensively described in the literature including in WO03102157A2, in Directed Mutagenesis, a Practical Approach, Ed. M. J.McPerson, IRL Press 1991. The method used here is the Kunkel method;this yields the stop template of the VL in plasmid pCES-Fab-h4D5-3ST.The stop template version of h4D5-VL is used as a template for theKunkel mutagenesis method (Kunkel et al. 1987, Methods in Enzymol.154:367-382), using mutagenic oligonucleotides designed tosimultaneously repair the stop codons and introduce mutations at thedesigned sites. Mutations in all three CDRs of the VL are introducedsimultaneously in a single mutagenesis reaction. This is extensivelydescribed in Sidhu et al. 2000, Methods Enzymol. 328:333-363. Themutagenesis reaction is electroporated into E. coli SS320 (Sidhu et al.,supra), and the transformed cells are grown overnight in the presence ofM13-VCS helper phage to produce phage particles that encapsulated thephagemid DNA and displayed Fab fragments on their surfaces. Methods forphage-display library manipulation, selection and screening of cloneshave been described in the literature, for example, see de Haard et al.,supra; Vajdos et al., supra and also the other examples). The resulting4D5-HYB1 library contains greater than 1×10⁸ unique members. This4D5-HVB1 library is selected twice on HER2 antigen as described inVajdos et al., supra, to yield a population of more than 65% ofantibodies with antigen-binding activity. These antibodies share theirVH region, but most carry different light chains The light chains ofthis population are obtained as ApaLI-AscI fragment (VLCL), and clonedas a pool into pCES-VH-2C4. This new library now contains a subset ofthe light chains of HYB1 that are likely to be compatible with antigenbinding in the context of h4D5. The library is selected once on antigen,and clones identified that mediate antigen binding. Light chains withidentical amino acid sequence and that mediate antigen-binding whenpaired with the h4D5-VH and with the 2C4-VH are identified by sequencinga panel of Ag-reactive clones from the selected h4D5-HYB1 library, andof Ag-reactive clones from the selected 2C4 sublibrary, and comparingthe sequences. Besides using antigen-reactivity in phage ELISA asreadout, the reactivity of the Fab fragments is tested in ELISA (asdescribed in de Haard et al., supra). This leads to the identificationof a panel of VLs that display are functionally pair with both VH-h4D5as well as VH-2C4. Within the panel the best VL is identified bydetermining the affinity of the interaction and the biological activityof the two respective Fab fragments. Methods for affinity determinationand biological activity of anti-HER2 Fabs are described in Kelley etal., 1992, supra, and Gerstner et al., 2002, supra, and are describedfurther below.

HYB2 library construction and screening of pairing-compatible VLs: TheHYB2-designed VL library contains 8000 variants only. Here a differentroute is followed to allow simultaneous expression, and detection ofantigen-binding variants, of h4D5 and 2C4 WI containing antibodies.First, the VL in pCES-Fab-h4D5 is mutated by Kunkel site-directedmutagenesis (Kunkel et al., supra) with Asparagine 30 changed to Serine(N30S), and Histidine 91 changed to Phenylalanine (H91F), according tothe design depicted in FIG. 23. Of the resulting clone, p4D5-VLmut,phage and Fab are produced and tested for binding in a dilutions seriesfor binding to Her-2 (extra-cellular domain) coated plate phage ELISA,to confirm that h4D5 maintains a minimal antigen-reactivity. Next astop-template version is made from this plasmid, by replacing one codonin the CDR2 of the VL with a TAA stop codon (residue 55, tyrosine ismutated from “tat” to “taa”; this residue is said to be required inorder to attain the antigen affinity of the humanized h4D5 antibody,Kelley et al., 1992, supra, thus will need to be fixed to “Y” to restorethe reading frame and antigen-binding), This stop template version ofthe light chain of h4D5v8 is cloned into pSCFV-3 (Example 2 and FIG.14B), by amplification of the VLCL region from the CDR2 stop-template.The design of the oligonucleotides used in this amplification is suchthat the whole VLCL segment is amplified and that after digestion thesegment can be directionally cloned for in frame expression of the lightchain under control of the arabinose promoter of pSCFV-3, and withoutany C-terminal tags. Briefly the VLCL is amplified with primers bindingto the 5′ and 3′ end of the cassette and at the 5′ providing a longoverhang in two PCR reactions to encode a region of approximately 90nucleotides encoding ribosome binding sites, start codon and bacterialleader sequence, to produce an EcoRV-EcoRI fragment that is cloned intothe PacI-EcoR1 sites bordering the third expression cassette in pSCFV-3.This plasmid, pVLmutST, is used as acceptor for the two heavy chains,after an internal BstEII site at position 143 of the insert was removed.The sequence of the final PacI-EcoR1 insert is given in SEQ ID NO:118.The heavy chains 2C4 and h4D5v8 are cloned in two steps as VH-CH1fragments into pSCFV-3 (FIG. 14B) to yield plasmid p2Fab-HER2 asindicated in FIG. 24. First the h4D5 VHCH1 region is amplified frompCES-WI-h4D5 and cloned as SfiI-BssHII fragment into pVLmutST. Thedesign of the primers is such that they, after cloning, arrangeappropriate reading frames with leaders and tags in pSCFV3, to yield thefinal junctional sequences as depicted in SEQ ID NO:119. Second, the 2C4heavy chain VHCH1 is amplified from pCES-VH-2C4 and cloned asBssHII-Notl fragment into this plasmid. Similarly, the design of theprimers is such that they, after cloning, arrange appropriate readingframes with leaders and tags in pSCFV3, to yield the final junctionalsequences as depicted in SEQ ID NO:119. This final plasmid, p2Fab-Her2,provides the expression of both heavy chain variable domains as Fdchains (linked to human gamma-1), and the expression of a yet stop codoncontaining light chain. The sequence of the HindIII-NotI and PacI-EcoRIinserts of p2Fab-HER2 is given in SEQ ID NOS:119 and 118, respectively.The heavy chains of the two humanized antibodies, h4D5-version 8 and2C4-version 574 are provided as fusions to the human CH1 domain, the Mycand VSV tag, respectively, and a HIS-tag for IMAC purification. Thelight chain in format VLCL is essentially derived from h4D5 but carriestwo designed VL mutations at positions 30 and 91, a stop codon in theCDR2, and has an internal BstEII site removed without amino acid change.

Plasmid p2Fab-HER2 is used as a template for the Kunkel mutagenesismethod (Kunkel et al. 1987, Methods in Enzymol. 154:367-382), usingmutagenic oligonucleotides designed to simultaneously repair the stopcodon in the VL-CDR2 and introduce mutations at the three designed sitesin CDR2, as indicated in FIG. 23. After electroporation and plating (asbefore), a small library of 50,000 clones is screened forpairing-compatible VL versions as follows. In the plasmid p2Fab-HER2,all three variable region genes are linked to a unique epitope tag thatprovides a way for their specific detection. Individual clones arepicked into 96-well plates (Nunc) and induced to express both heavychains and the one light chain, using conditions as described in Example4, with the exception that arabinose is also added as inducer at thesame time as the IPTG. The next day the supernatant of the cultures istested for the presence of HER2 reactive Fabs, in an ELISA essentiallyas in Example 4. Multiple assays are carried out with the same sample,using either anti-myc or anti-VSV secondary reagents to detect thepresence of the h4D5-Fab or the 2C4-Fab, respectively.

A dual-reactive clone designated 3-8E3, which binds HER-2 in ELISA withboth the anti-VSV and anti-Myc tag reagents, is chosen for furtheranalysis. The Fab mixture of this clone is expressed to 10-L scale leveland purified from E. coli Supernatants according to Kelley et al., 1992,supra, page 5435-5436. Briefly, the culture supernatant is microfilteredby tangential flow filtration, concentrated by ultrafiltration andfiltered over DEAE-Sepharose-FF. The antibody mixture in theflow-through fraction is subjected to affinity chromatography onProtein-G-Sepharose-FF. The Fab mixture is eluted with 0.1 M glycine, pH3.0. The total protein concentration is determined by A₂₈₀ measurementsusing an ∈₂₈₀ of 67 mM⁻¹ cm⁻¹.

The binding constant of individual Fabs or the apparent binding constantof the Fab mix are measured by ELISA essentially as described by Vajdoset al., 2002, supra, on page 426. Briefly, NUNC 96-well maxisorbimmunoplates are coated overnight at 4° C. with HER2-ECD (1 microgram/mlin 50 mM carbonate buffer, pH 9.6), and the plates blocked for one hourat room temperature with 0.5% BSA in PBS-0.05% TWEEN®-20. Serialdilutions of Fab protein are incubated on the HER2-ECD coated plates fortwo hours at room temperature, and the plates washed. Bound Fab isdetected with biotinylated murine anti-human kappa chain antibodyfollowing by streptavidin—horseradish peroxidase conjugate (Sigma),using 3,3′,5,5′-tetramethyl benzidine (TMB) as substrate (Kirsgaard andPerry Laboratories, Gaithersburg, Md.). The actual binding constant ofone Fab in the mixture of two Fabs is measured by replacing thebiotinylated murine anti-human kappa chain antibody of the above testwith biotinylated anti-MYC-tag (for h4D5) or biotinylated anti-VSV tag(for 2C4) antibodies (antibodies similar to those described in Example2). Titration curves are fit with a four-parameter non-linear regressioncurve-fitting program (KaledaGraph, Synergy Software) to determine theEC50 values, the Fab concentrations corresponding to half-maximalbinding signals. Examples for h4D5, 2C4 and the 3-8E3 mixture is givenin FIG. 25. The 3-8E3 mix is confirmed to contain two functional Fabantibody fragments, h4D5* and 2C4*, in which the * indicates that thelight chain variable region is different from the two original humanizedlight chains of h4D5 and 2C4 (in FIG. 24), The ratio of the two Fabantibodies that are present in the 3-8E3 mix is analyzed byelectrospray-ionization mass spectrometry essentially as described inKelley et al., 1992, supra. There is a difference in the molecularweights of the Fabs on the basis of the heavy chains of 2C4 and h4D5differing in approximately 68 dalton, well above the standard deviationof the assay (in the range of three to seven dalton).

The biological activity of the Fab mixtures is compared with that of theindividual monoclonal Fab fragments. The growth inhibitorycharacteristics are evaluated using the breast cancer cell line, SK-BR-3(see, Hudziak et al., 1989, Mol. Cell. Biol. 9:1165-1172), using theassay conditions described on page 50 of WO 0100245A2. An exemplarygraph in FIG. 25 shows the growth inhibition curves for h4D5 Fab andmixtures of 4D5* and 2C4* (see, Example 17) that utilize differentpairing-compatible light chains, indicated with VL1 to VL7. The Fabs arefurther evaluated for their ability to inhibit HRG-stimulated tyrosinephosphorylation of proteins in the Mr 180,000 range from whole-celllysates of MCR7 cells, which are known to express all known ErbBreceptors (as in WO 0100245A2, page 50-51). As a control, 2C4 as Fab isused; this antibody is very effective in disrupting the formation of thehigh affinity HER2/HER3 binding site on MCF7 cells.

Once the activity of the Fabs in the mixture confirmed, the selected,pairing-compatible VL of 3-8E3, is used to build an OLIGOCLONICS® of theIgG format, essentially as described in the previous Example 10. Thisresults in the production of 30 cell clones each producing a mixture ofthe bivalent h4D5* and 2C4* antibodies, and the bispecific combination;the IgGs are purified from the cell supernatants by protein A columnchromatography as described above, and the concentration of the totalIgG present in the mixtures determined. The biological activity of theresulting IgG-mixtures is tested as in Nahta et al., Cancer Research64:2343-2346 (2004), using a growth inhibition assay of BT474 breastcancer cells as described on page 2343 of this paper. Briefly BT474cells are treated in triplicate with two-fold serial dilutions of theIgG mixtures in the range of 0.1 to 25 ng/ml. After five days, cells aretrypsinized and counted by trypan blue exclusion. The growth inhibitionis calculated as the fraction of viable cells compared with untreatedcultures. As controls, the original antibodies hu4D5-v8 (trastuzumab)and 2C4 (Pertuzumab) are used, as well as a 1:1 mixture of thesemonoclonal antibodies. The mixture with the most synergic activitybetween the two binding sites is identified based on the dose-effectplots as described in the legend of FIG. 1 on page 2344 in Nahta et al.,2004, supra. Other tests to confirm the synergistic activity aredescribed in this paper (in vitro tests: apoptosis induction, Aktsignaling), in WO 0100245 A2 (in vitro tests and in vivo tests, such ashuman tumor xenograft models described in Examples 5 to 7 and in FIGS.10 to 13) and in Franklin et al., 2004, supra (in vitro HER2/HER3heterodimerization using COS7 transfected cells).

Other examples of antibodies that can be combined with one or both ofthese anti-ErbB2 antibodies are antibodies with pairing-compatiblechains that function as an anti-angiogenic agent (e.g., an anti-VEGFantibody); target the EGF-receptor (or ErbB1; e.g., C225 or ZD1839); orthat are anti-ErbB2 antibody that strongly induce apoptosis, such as 7C2or 7F3 (WO 0100245 A2). Pairing-compatible light chains are identifiedusing the methods described herein.

Example 18 Pairing-Compatible Antibodies to Produce a Mixture ofHepatocyte Growth Factor/Scatter Factor (HGF/SF)-Targeting Antibodiesthat Block Multiple Biological Activities

HGF/SF is a ligand that binds to the Met receptor tyrosine kinase.HGF/SF is composed of an a chain containing the N-terminal domain andfour kringle domains covalently di-sulfide linked to the β chain.Binding of HGF/SF to the Met receptor tyrosine kinase induces multiplebiological activities, including cell proliferation and cell invasion,and outgrowth of blood vessels (angiogenesis). In addition, binding ofHGF/SF to Met prevents programmed cell death (reviewed in C. Birchmeieret. al. Nat. Rev. Mol. Cell Biol. 4:915-925 (2004). The Met receptor isexpressed by many solid tumors and Met-HGF/SF signaling has been shownto be involved in tumor development, invasion and metastasis (J. M.Cherrington et al., Adv. Cancer. Res. 79:1-38 (2000); S. Rong et al.,Mol. Cell Biol. 12, 5152-5158 (1992).

Monoclonal antibodies against HGF/SF have been produced to study theircapacity to block the diverse biological activities of HGF/SC (B. Cao etal., Proc. Natl. Acad. Sci. U.S.A., 98, 7443-7448 2001). The antibodieswere produced by immunizing mice with human HGF/SF and generatinghybridomas secreting monoclonal antibodies. The polyclonal serum frommice immunized with HGF/SC showed potent neutralizing activity of allbiologic activities of HGF/SF. In contrast a large panel of monoclonalantibodies that bind to the human HGF/SCF was shown to lack the capacityto completely block all biological activities of HGF/SC (B. Cao et al.,Proc. Natl. Acad. Sci. USA, 98, 7443-7448 2001). Combinations of twoanti-HGF/SF monoclonal antibodies still lacked full blocking activitywhile several mixtures of three monoclonal antibodies potentlyneutralized all HGF/SF activity in in vitro assays. It was concludedthat blocking of the biological activities of HGF/SF requires thesimultaneous binding of multiple monoclonal antibodies against differentepitopes of the HGF/SF ligand (B. Cao et. al., Proc. Natl. Acad. Sci.USA, 98, 7443-7448 2001).

Mixtures of monoclonal antibodies directed against the same targetmolecule that block the complete spectrum of biological activities ofthe molecule are very valuable contributions to the therapeutic arsenal,especially when such blocking activities can not be achieved withmonoclonal antibodies. Production of such multiple antibodies in apharmaceutical manner and in a commercially viable way will become veryimportant. In this example, described is how mixtures of monoclonalantibodies against the HGF/SF ligand are isolated and used to constructan OLIGOCLONICS® that efficiently blocks all biological activities ofthis ligand.

Phage antibody scFv or Fab libraries that are formed by focusing thediversity in one variable region and keeping the other variable regioninvariable, for example a germ line sequence, are particularly relevant.From such libraries it is feasible to isolate antibodies with adifferent heavy chain yet identical light chain, or vice versa (FIG. 3).Such antibodies are readily reformatted into an OLIGOCLONICS® format. Inthe art, it has been described that antibodies that share the same VLgene but have different VH genes and widely varying specificities can beobtained from phage antibody display libraries (Nissim et al. (1994),EMBO J. 13:692-698). A sub-library of the semi-synthetic scFv library(de Kruif et al. (1995) J. Mol. Biol. 248:97) described in Example 7 isused to select antibodies against recombinant human HGF/SF.

The HGF/SF ligand is produced and purified from S-114 cells (NIH 3T3cells transformed with human HGF/SF and Met) as described (S. Rong etal. (1993) Cell Growth Differ. 4, 563-569). For phage selections,96-well plates are coated with 2.5 μg/ml HGF/SF in coating buffer (0.2 MNa₂CO₃/NaHCO₃, pH 9.6; 50 μl per well) overnight at 4° C. The plateswere then blocked with PBS containing 1% BSA (200 μ/well) overnight at4° C. Selections of phages binding to human HGF/SF are performed asdescribed in the previous examples. The binding of phages selected fromthe library is monitored by a HGF/SF ELISA using 96-well plates coatedwith 2.5 μg/ml HGF/SF in coating buffer (0.2 M Na₂CO₃/NaHCO₃, pH 9.6; 50μl per well) overnight at 4° C. The plates are then blocked with PBScontaining 1% BSA (200 μl/well) overnight at 4° C.

The VH regions from individual monoclonal antibodies and the single VLregion are cloned into the eukaryotic expression vector for humanmonoclonal antibodies as described in Example 10 and subsequentlyintroduced into eukaryotic CHO cells by transfection. For eachtransfection, the plasmids encoding two or more different VH regions aremixed in various ratios and used at a concentration of 1 to 10 μg/ml.Clones secreting human antibodies are generated essentially as describedin Example 10 and the supernatants monitored for HGF/SF-specificantibodies with an ELISA in 96-well plates coated with HGF/SF asdescribed in the previous paragraph. Supernatants from clones secretinganti-HGF/SF antibodies are used to determine the capacity of mixtures toblock the biological activities of HGF/SF.

Supernatants from transfectants are screened for neutralizing HGF/SFcapacity in the Madin-Darby canine kidney (MDCK) scatter assay asdescribed (B. Cao et. al., Proc. Natl. Acad. Sci. USA, 98, 7443-74482001). MDCK cells are plated at 7.5×10⁴ cells per 100 μl per well withor without HGF (5 ng/well) in DMEM with 5% FBS. Three hundredmicroliters of supernatants at two-fold serial dilutions is then addedto 96-well plates. A rabbit polyclonal-neutralizing antiserum (1μl/well; ref S. Koochekpour et. al. (1997) Cancer Res. 57, 5391-5398) isincluded as control. Following overnight incubation at 37° C., cells arethen stained with 0.5% crystal violet in 50% ethanol (vol/vol) for tenminutes at room temperature, and scattering is viewed using a lightmicroscope.

Supernatants from transfectants are also screened for neutralizingHGF/SF capacity in the Branching Morphogenesis Assay as described.Branching morphogenesis assay using SK-LMS-1 cells was conducted asdescribed (M. Jeffers et al. (1996) Mol. Cell. Biol. 16, 1115-1125).Briefly, cell suspensions are mixed with an equal volume of GFR-Matrigel(Becton Dickinson), plated at 5×10⁴ cells per 125 μl per well in a96-well culture plate, and incubated for 30 minutes at 37° C. HGF/SF,with or without neutralizing mAbs, is added along with DMEM containing10% FBS on top of the gel. After 72 to 96 hours of incubation at 37° C.,representative wells are photographed at ×400 magnification.

Example 19 Pairing-Compatible Antibodies to Produce a Mixture ofAntibodies that Block Vascular Endothelial Cell Growth Factor Receptor 1(VEGF-R1) and VEGF-R2

Vascular endothelial growth factor (VEGF) is a key regulator ofangiogenic processes during adult life such as wound healing, diabeticretinopathy, rheumatoid arthritis, psoriasis, inflammatory disorders andtumor growth and metastasis (N. Ferrara et. al., Curr Top. Microbiol.Immunol. 237-1-30 (1999); M. Klagsbrun et al., Cytokine Rev. 7, 259-270(1996); G. Neufeld et al. FASEB J. 13, 9-22 (1999)). VEGF binds to andmediates its activity mainly through two tyrosine kinase receptors,VEGF-R1 (also named Flt-1) and VEGF-R-2 (also named KDR). Numerousstudies have shown that overexpression of VEGF and its receptors plays arole in associated-associated angiogenesis and hence in tumor growth andmetastasis (J. Folkman, J. Nat. Med. 1, 27-31 (1995); Z. Zhu et. al.,Invest. New Drugs 17, 195-212 (1999)).

A human anti-VEGF monoclonal antibody binding to VEGF and blocking itsbinding to the VEGF-R1 has recently been approved by the FDA for thetreatment of patients with metastatic colorectal cancer(www.fda.gov/cder/foi/appletter/2004/1250851tr.pdf). This shows thatmonoclonal antibodies that block angiogenesis provide an important toolin the treatment of solid tumors.

In WO 04003211 A1, Zhu describes bispecific antibodies with one part ofthe molecule blocking the binding of VEGF to VEGF-R1 and another part ofthe molecule blocking binding of VEGF to VEGF-R2. In addition, thebi-specific antibody prevents the homodimerization of the VEGF receptorsand thus blocking VEGF-R-mediated cellular signaling Compared to bindingto a single VEGF-R, dual binding can result in more potent inhibition ofVEGF-stimulated cellular functions such as, for example, proliferationof endothelial cells. The bispecific antibodies described by Zhucomprise single chain Fv antibody fragments fused to the heavy and lightchain constant regions of an IgG molecule. Because of the nature of thebispecific molecules, they can be expected to be immunogenic uponinjection in humans, impeding their clinical effectiveness. Mixtures ofhuman antibodies as represented in the OLIGOCLONICS® format that blockboth the VEGF-Rl and VEGR-R2 while retaining optimal clinical efficacymay be an important addition to the arsenal of anti-solid tumor drugs.Such an OLIGOCLONICS® is obtained as follows:

The soluble fusion protein VEGF-R2 fused to alkaline phosphatase(VEGF-R2-AP) is expressed in stably-transfected NIH 3T3 cells andpurified from cell culture supernatant by affinity chromatography asdescribed (D. Lu et al., J. Biol. Chem. 275, 14321-14330 (2000)).VEGF-R1-Fc fusion protein is purchased from R&D Systems (Minneapolis,MN). VEGF-R2-AP is coated to Maxisorp Star tubes plates at aconcentration of 10 μg/ml and subsequently, the tubes are blocked with3% milk/PBS as described in WO 003211 and D. Lu et al., Cancer Res.61:7002-7008 (2001). The phage library used for selection of scFvantibody fragments specific for VEGF-R2 contains a single light chainand is diversified in the heavy chain as described in the previousExample 7. Selection of phages is carried out as described in theprevious examples. The specificity of selected scFv antibody fragmentsis determined in ELISA with 10 μg/ml VEGF-R2-AP coated to Maxisorp96-well plates and scFv binding, washing and detection steps asdescribed in the previous examples. As a control for binding to the APmoiety, scFv are assayed for binding to a control AP fusion proteinssuch as ELF2-AP (GenHunter Corp., Nashville, Tenn.). Selection of phagesbinding to the VEGF-R1 is carried out by coating Maxisorp Star tubeswith 10 μg/ml VEGF-R1-Fc and performing rounds of selection as describedin the previous examples. The specificity of selected scFv is analyzedin ELISA with 10 μg/ml VEGF-R1-Fc coated to 96-well plates. As a controlfor binding to the Fc portion VEGF-R1-Fc, plates are coated with the Fcfusion protein rhsThy-1:Fc (product number ALX-203-004, AlexisBiochemicals, Lausen, Switzerland).

The VH regions from individual monoclonal antibody fragments and thesingle VL region are cloned into the eukaryotic expression vector forhuman monoclonal antibodies as described in Example 10 and subsequentlyintroduced into eukaryotic CHO cells by transfection. For eachtransfection, the plasmids encoding two or more different VH regions aremixed in various ratios and used at a concentration of 1 to 10 μg/ml.Clones secreting human antibodies are generated essentially as describedin Example 10 and the supernatants monitored for VEGF-Rl andVEGF-R2-specific antibodies with an ELISA in 96-well plates coated withVEGF-R1-Fc and VEGF-R2-AP as described in the previous paragraph, andusing secondary antibodies that specifically bind to the humanantibodies. Supernatants from clones secreting antibodies to bothreceptors are used to determine the biological activity of the mixturesin VEGF-Rl and VEGF-R2 blocking assays and in an anti-mitotic andleukemia migration assays.

VEGF-R1 and VEGF-R2 blocking assays are performed as described (Z. Zhuet al., Cancer Res. 58:3209-14 (1998); D. Lu et al., J. Immunol.Methods, 230:159-71 (1999). The anti-mitotic and leukemia migrationassays are performed as described in WO 04003211 A1. To measure whetherthese antibody mixtures compete with VEGF for binding to the receptors,assays are carried out that measure the level of antibody-inducedinhibition of VEGF-associated effects. For example, the effect of theantibody cocktail on VEGF-induced endothelial cell proliferation ismeasured using a thymidine incorporation assay. Numerous in vitro and invivo assays have been described to measure the effect of ligandsinterfering with the VEGF - VEGF-receptor interaction. Some suitableassays are described in Gerbert et al., J Biol. Chem. 1998, 273:30336(cell survival assay, endothelial cell apoptosis, Akt phosphorylationassay, as on page 30337); in Mendel et al., Clin. Cancer Res. 2000,6:4848-4858 (s.c. xenograft model in athymic mice, surface expression ofKDR, ¹²⁵I VEGF binding assay, and Flk-1 receptor kinase assay, as onpages 4849-4850). These and other suitable assays are reviewed inAuerbach et al., 2003, Clin. Chemistry 49(1):32-40.

Example 20 Human Light Chain V-Gene Clones

This Example describes the rationale behind the choice of two humanlight chain V- genes, one gene of the kappa type and one gene of thelambda type, that are used as a proof of concept for light chainexpressing transgenic mice. De Wildt et al. 1999 (de Wildt et al.(1999), J. Mol. Biol. 285(3):895) analyzed the expression of human lightchains in peripheral IgG-positive B-cells. Based on these data, IGKV1-39(O12) and IGLV2-14 (2a2) were chosen as light chains as they were wellrepresented in the B-cell repertoire. The J-segment sequence of thelight chains has been chosen based upon sequences as presented inGenBank ABA26122 for IGKV1-39 (B. J. Rabquer, S. L. Smithson, A. K.Shriner and M. A. J. Westerink) and GenBank AAF20450 for IGLV2-14 (O.Ignatovich, I. M. Tomlinson, A. V. Popov, M. Bruggemann and G. J.Winter, J. Mol. Biol. 294 (2):457-465 (1999)).

All framework segments are converted into germline amino acid sequencesto provide the lowest immunogenicity possible in potential clinicalapplications.

Example 21 Obtaining Mouse Heavy Chain V-genes that Pair with HumanIgkv1-39 Gene Segment to Form Functional Antibody Binding Sites

This example describes the identification of mouse heavy chain V-genesthat are capable of pairing with a single, rearranged human germlineIGKV1-39/J region. A spleen VH repertoire from mice that were immunizedwith tetanus toxoid was cloned in a phage display Fab vector with asingle human IGKV1-39-C kappa light chain and subjected to panningagainst tetanus toxoid. Clones obtained after a single round of panningwere analyzed for their binding specificity. The murine VH genesencoding tetanus toxoid-specific Fab fragments were subjected tosequence analysis to identify unique clones and assign VH, DH and JHutilization.

Many of the protocols described here are standard protocols for theconstruction of phage display libraries and the panning of phages forbinding to an antigen of interest and described in Antibody PhageDisplay: Methods and Protocols (editor(s): Philippa M. O′Brien andRobert Aitken).

Immunizations: BALB/c mice received one immunization with tetanus toxoidand were boosted after six weeks with tetanus toxoid.

Splenocyte isolation: Preparation of spleen cell suspension. Afterdissection, the spleen was washed with PBS and transferred to a 60 mmPetri dish with 20 ml PBS. A syringe capped with 20 ml PBS and a G20needle was used to repeatedly flush the spleen. After washing theflushed cells with PBS, the cells were carefully brought into suspensionusing 20 ml PBS and left on a bench for five minutes to separate thesplenocytes from the debris and cell clusters. The splenocytessuspension was transferred on top of a FICOLL-PAQUE® PLUS-filled tubeand processed according to the manufacturer's procedures for lymphocyteisolation (Amersham Biosciences).

RNA isolation and cDNA synthesis: After isolation and pelleting oflymphocytes, the cells were suspended in TRIzol LS Reagent (Invitrogen)for the isolation of total RNA according to the accompanyingmanufacturer's protocol and subjected to reverse transcription reactionusing 1 microgram of RNA, Superscript III RT in combination with dT20according to manufacturer' s procedures (Invitrogen).

PCR amplification of cDNA: The cDNA was amplified in a PCR reactionusing primer combinations that allow the amplification of approximately110 different murine V-genes belonging to 15 VH families (Table 1;RefSeq NG_005838; Thiebe et al. 1999, European Journal of Immunology29:2072-2081). In the first round, primer combinations that bind to the5′ end of the V-genes and 3′ end of the J regions were used. In thesecond round, PCR products that were generated with the MJH-Rev2 primerwere amplified in order to introduce modifications in the 3′ region toenable efficient cloning of the products. In the last round ofamplification, all PCR products were amplified using primers thatintroduce a SfiI restriction site at the 5′ end and a BstEII restrictionsite at the 3′ end (see, FIGS. 26 and 27, and Table 1).

Reaction conditions for 1st round PCR: four different reactionscombining all 25 forward primers (MVH1 to MVH25, Table 1 and FIG. 27)and one reverse primer per reaction (MJH-Rev1, MJH-Rev2, MJH-Rev3 orMJH-Rev4; see Table 1 and FIG. 32). Fifty microliters PCR volumes werecomposed of 2 microliters cDNA (from RT reactions), 10 microliters 5*Phusion polymerase HF buffer, 40 nM of each of the 25 forward primers(total concentration of 1 micromolar), 1 micromolar reverse primer, 1microliter 10 mM dNTP stock, 1.25 unit Phusion polymerase and sterile MQwater. The thermocycler program consisted of a touch down program: onecycle 98° C. for 30 seconds, 30 cycles 98° C. for ten seconds, 58° C.decreasing 0.2° C. per cycle ten seconds, 72° C. 20 seconds and onecycle 72° C. for three minutes. The second round PCR program was set uponly for the products of the first PCR that contain the MJH-Rev2 primer:two different reactions combining either the ExtMVH-1 or ExtMVH-2primers (Table 1 and FIG. 27) in combination with the reverse primerExtMJH-Rev2int (Table 1 and FIG. 27). Fifty microliters PCR volumes werecomposed of 50 ng PCR product (from first PCR round), 10 microliters 5*Phusion polymerase HF buffer, 500 nM of each forward primer, 1micromolar reverse primer, 1 microliter 10 mM dNTP stock, 1.25 unitPhusion polymerase and sterile MQ water. The thermocycler programconsisted of a touch down program followed by a regular amplificationstep: one cycle 98° C. for 30 seconds, ten cycles 98° C. for tenseconds, 65° C. decreasing 1.5° C. per cycle ten seconds, 72° C. 20seconds, ten cycles 98° C. for ten seconds, 55° C. ten seconds, 72° C.20 seconds and one cycle 72° C. for three minutes. The third round PCRprogram was setup as described in FIG. 27. Fifty microliters PCR volumeswere composed of 50 ng PCR product (from earlier PCR rounds, FIG. 27),10 microliters 5* Phusion polymerase HF buffer, 1 micromolar forwardprimer (Table 1 and FIG. 27), 1 micromolar reverse primer, 1 microliter10 mM dNTP stock, 1.25 unit Phusion polymerase and sterile MQ water. Theprogram consists of a touch down program followed by a regularamplification step: one cycle 98° C. for 30 seconds, ten cycles 98° C.for ten seconds, 65° C. decreasing 1.5° C. per cycle ten seconds, 72° C.20 seconds, ten cycles 98° C. for ten seconds, 55° C. ten seconds, 72°C. 20 seconds and one cycle 72° C. for three minutes. After PCRamplifications, all PCR products were gel purified using Qiaex IIaccording to the manufacturer's protocols.

Restriction enzyme digestions: Purified products were digested withBstEII and SfiI in two steps. First 1 microgram of DNA was digested in100 microliters reactions consisting of 10 microliters of 10* NEB buffer3 (New England Biolabs), 1 microliter 100* BSA, 12.5 unit BstEII andsterile water for six hours at 60° C. in a stove. The products werepurified using Qiaquick PCR Purification kit from Qiagen according tothe manual instructions and eluted in 40 microliters water. Next, allproducts were further digested with SfiI in 100 microliters reactionsconsisting of 10 microliters of 10* NEB buffer 2 (New England Biolabs),1 microliter 100* BSA, 12.5 unit SfiI and sterile water for 12 hours at50° C. in a stove. The digested fragments were purified by Qiaquick GelExtraction kit following gel separation on a 20 cm 1.5% agarose TBE plusethidium bromide gel at 80 V. 100 micrograms of the acceptor vector(MV1043, FIGS. 28 and 27) was digested with 50 units Eco91I in 600microliters under standard conditions (Tango buffer) and next purifiedon a 0.9% agarose gel. After a second digestion step under prescribedconditions with 400 units SfiI in 500 microliters for 12 hours, 100units BsrGI were added for three hours at 50° C.

Ligations: Each PCR product was ligated separately according to thefollowing scheme: 70 ng digested PCR products, 300 ng digested acceptorvector, 100 units T4 Ligase (NEB), 1* ligase buffer in 30 microlitersfor 16 hours at 12° C. The ligation reactions were purified withphenol/chloroform/isoamyl alcohol extractions followed by glycogenprecipitations (Sigma Aldrich #G1767) according to the manufacturer'sprotocol and finally dissolved in 25 microliters sterile water.

Transformations and library storage: The purified ligation products weretransformed by electroporation using 1200 microliters TG1electrocompetent bacteria (Stratagene #200123) per ligation batch andplated on LB carbenicillin plates containing 4% glucose. Libraries wereharvested by scraping the bacteria in 50 ml LB carbenicillin. Aftercentrifugation at 2000 g for 20 minutes at 4° C., the bacterial pelletswere resuspended carefully in 2 ml ice cold 2*TY/30% glycerol on icewater and frozen on dry ice/ethanol before storage at −80° C.

Library amplification: Libraries were grown and harvested according toprocedures as described by Kramer et al. 2003 (Kramer et al. (2003),Nucleic Acids Res. 31(11):e59) using VCSM13 (Stratagene) as helper phagestrain.

Selection of phages on coated immunotubes: Tetanus toxoid was dissolvedin PBS in a concentration of 2 μg/ml and coated to MaxiSorp Nunc-ImmunoTube (Nunc 444474) overnight at 4° C. After discarding the coatingsolution, the tubes were blocked with 2% skim milk (ELK) in PBS(blocking buffer) for one hour at RT. In parallel, 0.5 ml of the phagelibrary was mixed with 1 ml blocking buffer and incubated for 20 minutesat room temperature. After blocking the phages, the phage solution wasadded to the tetanus toxoid-coated tubes and incubated for two hours atRT on a slowly rotating platform to allow binding. Next, the tubes werewashed ten times with PBS/0.05% TWEEN®-20 followed by phage elution byan incubation with 1 ml 50 mM glycine-HCl pH 2.2 ten minutes at RT onrotating wheel and directly followed by neutralization of the harvestedeluent with 0.5 ml 1 M Tris-HCl pH 7.5.

Harvesting phage clones: Five ml XL1-Blue MRF (Stratagene) culture atO.D. 0.4 was added to the harvested phage solution and incubated for 30minutes at 37° C. without shaking to allow infection of the phages.Bacteria were plated on Carbenicillin/Tetracycline 4% glucose 2*TYplates and grown overnight at 37° C.

Phage production: Phages were grown and processed as described by Krameret al. 2003 (Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) usingVCSM13 as helper phage strain.

Phage ELISA: ELISA plates were coated with 100 microliters tetanustoxoid per well at a concentration of 2 micrograms/ml in PBS overnightat 4° C. Plates coated with 100 microliters thyroglobulin at aconcentration of 2 micrograms/ml in PBS were used as a negative control.Wells were emptied, dried by tapping on a paper towel, filled completelywith PBS-4% skimmed milk (ELK) and incubated for one hour at roomtemperature to block the wells. After discarding the block solution,phage minipreps pre-mixed with 50 μl blocking solution were added andincubated for one hour at RT. Next five washing steps with PBS-0.05%TWEEN®-20 removed unbound phages. Bound phages were detected byincubating the wells with 100 microliters anti-M13-HRP antibodyconjugate (diluted 1/5000 in blocking buffer) for one hour at roomtemperature. Free antibody was removed by repeating the washing steps asdescribed above, followed by TMB substrate incubation until colordevelopment was visible. The reaction was stopped by adding 100microliters of 2 M H₂SO₄ per well and analyzed on an ELISA reader at 450nm emission wavelength (Table 2). Higher numbers indicate strongersignals and thus higher incidence of specific binding of the phage-Fabcomplex.

Sequencing: Clones that gave signals at least three times above thebackground signal (Table 2) were propagated, used for DNA miniprepprocedures (see, procedures Qiagen miniPrep manual) and subjected tonucleotide sequence analysis. Sequencing was performed according to theBig Dye 1.1 kit accompanying manual (Applied Biosystems) using a reverseprimer (CH1_Rev1, Table 1) recognizing a 5′ sequence of the CH1 regionof the human IgG1 heavy chain (present in the Fab display vector MV1043,FIGS. 28 and 37A-37Z). Mouse VH sequences of 28 tetanus toxoid bindingclones are depicted in Table 3. The results show that the selectedmurine VH genes belong to different gene families, and differentindividual members from these gene families are able to pair with therearranged human IGKV1-39/J VH region to form functional tetanustoxoid-specific antibody binding sites. From the sequence analyses, itwas concluded that the murine VH regions utilize a diversity of DH andJH gene segments.

Example 27 Silencing of the Mouse Kappa Light Chain Locus

This example describes the silencing of the mouse endogenous kappa lightchain locus. The endogenous kappa locus is modified by homologousrecombination in ES cells, followed by the introduction of geneticallymodified ES cells in mouse embryos to obtain genetically adaptedoffspring.

A vector that contains an assembled nucleotide sequence consisting of apart comprising the J-region to 338 bp downstream of the J5 gene segmentfused to a sequence ending 3′ of the 3′ CK enhancer is used forhomologous recombination in ES cells. The assembled sequence is used todelete a genomic DNA fragment spanning from 3′ of the JK region to just3′ of the 3′ CK enhancer. As a consequence of this procedure, the CKconstant gene, the 3′ enhancer and some intergenic regions are removed(see, FIGS. 29, 43A and 43B).

Construction of the targeting vector: A vector that received 4.5-8 kbflanking arms on the 3′ and 5′ end fused to the deletion segment wasused for targeted homologous recombination in an ES cell line. Both armswere obtained by PCR means ensuring maximum homology. The targetingstrategy allows generation of constitutive KO allele. The mouse genomicsequence encompassing the Igk intronic enhancer, Igk constant region andthe Igk 3′ enhancer was replaced with a PuroR cassette, which wasflanked by F3 sites and inserted downstream of the Jk elements.Flp-mediated removal of the selection marker resulted in a constitutiveKO allele. The replacement of the Igk MiEk-Igk C-Igk 3′E genomic region(approximately 10 kb) with a F3-Puro cassette (approx. 3 kb) was likelyto decrease the efficiency of homologous recombination. Therefore, thearms of homology were extended accordingly and more ES cell colonieswere analyzed after transfection in order to identify homologousrecombinant clones.

Generation of ES cells bearing the deleted kappa fragment: Thegeneration of genetically modified ES cells was essentially performed asdescribed (Seibler et al. (2003), Nucleic Acids Res. February 15;31(4):e12). See also Example 33 for a detailed description.

Generation of ES mice by tetraploid embryo complementation: Theproduction of mice by tetraploid embryo complementation usinggenetically modified ES cells was essentially performed as described(Eggan et al., PNAS 98:6209-6214; J. Seibler et al. (2003), NucleicAcids Res. February 15; 31(4):e12; Hogan et al. (1994), Summary of mousedevelopment, Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor N.Y., pp. 253-289).

Example 23 Silencing of the Mouse Lambda Light Chain Locus

This Example describes the silencing of the mouse endogenous lambdalight chain locus. The endogenous lambda locus is modified by homologousrecombination in ES cells followed by the introduction of geneticallymodified ES cells in mouse embryos to obtain genetically adaptedoffspring.

Two regions of the murine lambda locus that together contain allfunctional lambda V regions are subject to deletion.

The first region targeted for homologous recombination-based deletion isa region that is located 408 bp upstream of the start site of the IGLV2gene segment and ends 215 bp downstream of IGLV3 gene segment, includingthe intergenic sequence stretch between these IGLV gene segments. Thesecond region that is subject to a deletion involves the IGLV1 genesegment consisting of a fragment spanning from 392 bp upstream to 171 bpdownstream of the IGLV1 gene segment. As a consequence of these twodeletion steps, all functional V-lambda genes segments are deleted,rendering the locus functionally inactive (FIGS. 30, 44A and 44B).

Construction of the Targeting Vectors

Vectors that received 3-9.6 kb flanking arms on the 3′ and 5′ end fusedto the deletion segment were used for targeted homologous recombinationin an ES cell line. Both arms were obtained by PCR means ensuringmaximum homology. In a first step, the mouse genomic sequenceencompassing the Igl V2-V3 regions were replaced with a PuroR cassetteflanked by F3 sites, which yields a constitutive KO allele afterFlp-mediated removal of selection marker (see, FIG. 44A). In a secondstep, the mouse genomic sequence encompassing the Igl V1 region wasreplaced with a Neo cassette in ES cell clones which already carried adeletion of the Igl V2-V3 regions (see, FIG. 44B). The selection marker(NeoR) was flanked by FRT sites. A constitutive KO allele was obtainedafter Flp-mediated removal of selection markers.

Generation of ES Cells Bearing the Deleted Lambda Fragment

The generation of genetically modified ES cells was essentiallyperformed as described (J. Seibler, B. Zevnik, B. Küter-Luks, S.Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N. Faust, G.Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kühn, F. Schwenk(2003), Nucleic Acids Res., February 15; 31(4):e12). See also, Example33 for a detailed description. To show that both targeting eventsoccurred on the same chromosome several double targeted clones wereselected for the in vitro deletion with pCMV C31deltaCpG. The cloneswere expanded under antibiotic pressure on a mitotically inactivatedfeeder layer comprised of mouse embryonic fibroblasts in DMEM HighGlucose medium containing 20% FCS (PAN) and 1200 μ/mL LeukemiaInhibitory Factor (Millipore ESG 1107). 1×10′ cells from each clone wereelectroporated with 20 μg of circular pCMV C31deltaCpG at 240 V and 500μF and plated on four 10 cm dishes each. Two to three days afterelectroporation, cells were harvested and analyzed by PCR. Primers usedwere:

2005_5: (SEQ ID NO: 1) CCCTTTCCAATCTTTATGGG 2005_7: (SEQ ID NO: 2)AGGTGGATTGGTGTCTTTTTCTC 2005_9: (SEQ ID NO: 3) GTCATGTCGGCGACCCTACGCC

PCR reactions were performed in mixtures comprising 5 μl PCR Buffer 10×(Invitrogen), 2 μl MgCl₂ (50 mM), 1 μl dNTPs (10 mM), 1μl first primer(5 04), 1μl second primer (5 μM), 0.4 μl Taq (5 U/ul, Invitrogen), 37.6μl H₂O, and 2 μl DNA. The program used was 95° C. for five minutes;followed by 35 cycles of 95° C. for 30 seconds; 60° C. for 30 seconds;72° C. for 1 minute; followed by 72° C. for ten minutes.

Generation of ES mice by tetraploid embryo complementation: Theproduction of mice by tetraploid embryo complementation usinggenetically modified ES cells was essentially performed as described(Eggan et al., PNAS 98:6209-6214; J. Seibler, B. Zevnik, B. Küter-Luks,S. Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N. Faust, G.Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kühn, and F. Schwenk(2003), Nucleic Acids Res., February 15; 31(4):e12; Hogan et al. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y.), pp. 253-289).

Example 24 Construction of the CAGGS Expression Insert Based on aRearranged Human Germline IGKV1-39/J-Ck gene (IGKV1-39/J-Ck)

This example describes the construction of a CAGGS expression cassetteincorporating the rearranged human germline IGKV1-39/J region. Thisinsert expression cassette encompasses cloning sites, a Kozak sequence,a leader sequence containing an intron, an open reading frame of therearranged IGKV1-39 region, a rat CK constant region from allele a and atranslational stop sequence (IGKV1-39/J-Ck; FIG. 31). The primaryconstruct consists of naturally occurring sequences and has beenanalyzed and optimized by removing undesired cis acting elements likeinternal TATA-boxes, poly adenylation signals, chi-sites, ribosomalentry sites, AT-rich or GC-rich sequence stretches, ARE-, INS- and CRSsequence elements, repeat sequences, RNA secondary structures, (cryptic)splice donor and acceptor sites and splice branch points (GeneArt GmbH).In addition, the codon usage in the open reading frame regions isoptimized for expression in mice. The intron sequence is unchanged andthus represents the sequence identical to the coding part of the humanIGKV1-39 leader intron.

At the 5′ end of the expression cassette, a Notl site was introduced andon the 3′ site a Nhel site. Both sites are used for cloning in the CAGGSexpression module. After gene assembly according to methods used byGeneArt, the insert is digested with Notl-Nhel and cloned into theexpression module containing a CAGGS promoter, a stopper sequenceflanked by LoxP sites (“foxed”), a polyadenylation signal sequence and,at the 5′ and 3′ end, sequences to facilitate homologous recombinationinto the Rosa26 locus of mouse ES cell lines. Promoter and/or cDNAfragments were amplified by PCR, confirmed by sequencing and/or cloneddirectly from delivered plasmids into an RMCE exchange vector harboringthe indicated features. A schematic drawing and the confirmed sequenceof the final targeting vector pCAGGS-IgVK1-39 are shown in FIGS.38A-38B-4. The targeting strategy is depicted in FIG. 38C.

Example 25 CAGGS Expression Insert Based on the Rearranged GermlineIGLV2-143 V Lambda Region (IGLV2-14/J-Ck)

This example describes the sequence and insertion of an expressioncassette incorporating the rearranged germline IGLV2-14/J V lambdaregion. This insert encompasses cloning sites, a Kozak sequence, aleader sequence containing an intron, an open reading frame of therearranged IGLV2-14/J region, a rat CK constant region from allele a anda translational stop sequence (IGLV2-14/J-Ck; FIG. 32). The primaryconstruct consists of naturally-occurring sequences and has beenanalyzed and optimized by removing undesired cis acting elements like:internal TATA-boxes, poly adenylation signals, chi-sites, ribosomalentry sites, AT-rich or GC-rich sequence stretches, ARE-, INS- and CRSsequence elements, repeat sequences, RNA secondary structures, (cryptic)splice donor and acceptor sites and splice branch points (GeneArt GmbH).In addition, the codon usage in the open reading frame regions wasoptimized for expression in mice. The intron sequence is unchanged andthus represents the sequence identical to the human IGKV1-39 leaderintron.

At the 5′ end of the expression cassette, a Notl site was introduced andon the 3′ site a NheI site. Both sites are used for cloning in the CAGGSexpression module as described by TaconicArtemis. After gene assemblyaccording to methods used by GeneArt, the insert was digested withNotI-NheI and cloned into the expression module containing a CAGGSpromoter, a stopper sequence flanked by LoxP sites (“foxed”), apolyadenylation signal sequence and, at the 5′ and 3′ end, sequences tofacilitate homologous recombination into the Rosa26 locus of mouse EScell lines. To construct the final ROSA26 RMCE targeting vector,promoter and/or cDNA fragments were amplified by PCR. Amplified productswere confirmed by sequencing and/or cloned directly from deliveredplasmids into an RMCE exchange vector harboring the indicated features.A schematic drawing and the confirmed sequence of the final targetingvector pCAGGS-IgVL2-14 is shown in FIGS. 40A-40B-4. The targetingstrategy is depicted in FIG. 40C.

Example 26 Expression of IGKV1-39/J-Ck in HEK293 cell lines(pSELECT-IGKV1-39/J-Ck)

This example describes a method to verify that the IGKV1-39/J-Ckconstructs described in Example 24 enable expression and detection ofthe IGKV1-39/J-Ck L chain in HEK293 cells. The IGKV1-39/J insert (FIG.31) was modified at the 5′ end by changing the Notl site into a Sallsite. This change is required for cloning of the product into theexpression cassette plasmid pSELECT-hygro (InvivoGen). The CAGGSexpression insert IGKV1-39/J-Ck and pSELECT-hygro were digested withSalI and NheI, ligated and used to transform competent XL1-Blue cellsusing standard techniques. Colonies were picked and DNA purified usingQiagen Midi-prep columns according to the manufacturer's procedures. Theresulting light chain (LC) expressing vector named0817676_pSELECT_0815426 was used to transfect HEK293 cells with Fugene6(Roche) according to the manufacturer's protocols. Supernatants werescreened for the presence of IGKV1-39/J-Ck light chains by ELISA andwestern blot using anti-rat-Ck antibodies (Beckton Dickinson #550336 and553871) and protocols used in the art.

The VH of anti-tetanus toxoid (TT) IgG MG1494 was cloned into IgGexpression vector MV1056 using restriction sites SfiI and BstEII. Theresulting clone was sequence verified. HEK293T cells were transfectedwith five different vector combinations as shown in Table 4 (see,Example 27 for details of vector 0817678_pSELECT_0815427). Supernatantswere harvested and IgG concentrations determined (see, Table 4). No IgGcould be detected for supernatants A and B containing light chain onlyas expected (detection antibody recognized Fc part of IgG). IgGconcentration in supernatants C and D was comparable to that of positivecontrol supernatant E, indicating correct expression of the light chainconstructs.

Binding to TT was analyzed by ELISA to check functionality of theproduced antibodies, using hemoglobin as negative control antigen. NoTT-specific binding could be detected for supernatants A and Bcontaining light chain only, as expected. TT-specific binding forsupernatants C and D was at least as good as for positive controlsupernatant E, confirming correct expression of the light chainconstructs and functional assembly with heavy chain Antibodies weredetected not only using an anti-human IgG secondary antibody, but alsoan anti-rat Ckappa light chain secondary antibody. The results confirmthat the anti-rat Ckappa antibody (BD Pharmingen #553871, clone MRK-1)recognizes the light chain expressed by the pSELECT vectors.

Supernatants were analyzed by non-reducing SDS-PAGE and Western blot(not shown). Detection using an anti-human IgG heavy chain antibody didnot show bands for supernatants A and B containing light chain only, asexpected. Results for supernatants C and D were comparable to positivecontrol supernatant E, with a band close to the 170 kD marker asexpected for intact IgG. Additional lower molecular weight bands wereobserved as well for supernatants C, D and E, which might representdegradation products, IgG fragments resulting from (partial) reductionand/or irrelevant protein bands due to non-specific binding of thedetection antibody.

Detection using an anti-rat Ckappa light chain antibody showed a bandclose to the 26 kD marker for supernatants A and B, as expected forlight chain only. This band was much more intense for A compared to B,indicating that the free IGKV1-39 light chain may be better expressedand/or more stable than the free IGLV2-14 light chain No bands weredetected for control supernatant E as expected, since the expressed IgGcontains a human Ckappa light chain For supernatants C and D, expectedbands close to the 170 kD marker were observed; lower molecular weightbands were also observed, but to a lesser extent than above using theanti-human IgG antibody.

In conclusion, transfection of the light chain expression constructscombined with the heavy chain of anti-tetanus toxoid (TT) IgG MG1494resulted in IgG production comparable to the positive control constructfor both the pSELECT kappa and lambda light chain constructs. Both IgGproductions yielded ELISA signals in a TT ELISA that were better than orcomparable to the control IgG. SDS-PAGE and Western blot analysisconfirmed the presence of intact IgG. The tested anti-rat Ckappaantibody worked efficiently in both ELISA and Western blot. Culturesupernatant from cells transfected with light chain constructs only didnot result in detectable IgG production nor in detectable TT-specificbinding, while free light chain was detected on Western blot.

Example 27 Expression of IGLV2-14/J-Ck in HEK293 cell lines(pSELECT-IGLV2-14/J-Ck)

This Example describes a method to verify that the IGLV2-14/J constructsdescribed in Example 25 enable expression and detection of theIGLV2-14/J-Ck L chain in HEK293 cells. The IGLV2-14/J-Ck insert (FIG.32) was modified at the 5′ end by changing the Notl site into a Sallsite. This change is required for cloning of the product into theexpression cassette plasmid pSELECT-hygro (InvivoGen). The CAGGSexpression insert IGLV2-14/J-Ck and pSELECT-hygro were digested withSalI and NheI ligated and used to transform competentXL1-Blue cellsusing standard techniques. Colonies were picked and DNA purified usingQiagen Midi-prep columns according to the manufacturer's procedures. Theresulting light chain (LC) expressing vector named0817678pSELECT_0815427 was used to transfect HEK293 cells with Fugene6(Roche) according to the manufacturer's protocols. Supernatants werescreened for the presence of IGLV2-14/J-Ck light chains by ELISA andwestern blot using anti-rat-Ck antibodies (Becton Dickinson #550336 and553871) and protocols used in the art. See Example 26 for details andresults.

Example 28 Construction of a VK Promoter-Driven Expression ConstructContaining an IGKV1-39/J Insert and Multiple Enhancer Elements Derivedfrom the Murine CK locus (VkP-IGKV1-39/J-Ck; VkP-012)

This example describes the construction of an expression cassette thatcontains relevant elements to enable B-cell anddevelopmental/differentiation stage-specific expression of therearranged human IGKV1-39 VK region, based on the IGKV1-39 VK promoterregion, leader containing an intron, germline V-gene, CDR3, IGKJsegment, mouse intergenic region located between Jk and CK, rat Ckallele a open reading frame, and a mouse intergenic fragment from the 3′end of the mouse CK gene ending just 3′ of the 3′ CK enhancer.

Optimized open reading frames of the leader, IGKV1-39 rearranged gene,and rat CK allele a gene, as described in Example 24, was used for theconstruction of the expression cassette. The VK promoter region wasobtained by gene synthesis procedures (GeneArt, GmbH) and is almostidentical to the sequence of the human IGKV1-39 region between −500 bpand the ATG (start site) of the gene. The only deviation from thenatural sequence is the introduction of a GCCACCATGG Kozak sequence (SEQID NO:102) at the ATG (start) site in order to promote translation. Agenomic fragment from a mouse BAC clone (TaconicArtemis) is used as thebasis for the introduction of individual elements. This fragment isidentical to the sequence of the mouse VK locus starting with the introndonor site located directly 3′ of the JK5 region and ending just 3′ ofthe 3′ CK enhancer and covers approximately 12.5 kb.

The final construct contains from 5′ to 3′ end the following elements:human genomic IGKV1-39 promoter (500 bp), a Kozak sequence, a humanIGKV1-39 leader part 1 (optimized), a human IGKV1-39 leader intron, ahuman IGKV1-39 leader part 2 (optimized), a human IGKV1-39 germline gene(optimized), a human J-region (optimized), a mouse intergenic regionincluding the intron enhancer element, a rat (Rattus norvegicus) kappaconstant region (optimized), and a mouse intergenic region including the3′ kappa enhancer. The elements of this expression cassette are shown inFIG. 33 and named VkP-IGKV1-39/J-Ck (VkP-O12). An outline of thepVkP-O12 vector and the targeting strategy is depicted in FIGS. 45A and46A. The vector was introduced into ES cells following standardprocedures (see, Example 33).

Example 29 Construction of a VK Promoter-driven Expression ConstructContaining An Iglv2-14/J Clone and Multiple CK Locus-Derived EnhancerElements (VkP-IGLVL2-14/J-Ck; VkP-2a2)

This example describes the same construct as described in Example 28,except that the IGKV1-39 gene and J-region are replaced by the optimizedhuman IGLV2-14 germline gene including a unique V-J region(VkP-IGLV2-14/J-Ck; VkP-2a2; FIG. 34).

Example 30 Construction of a VK Promoter-driven Expression ConstructContaining An Igkv1-39 Clone Lacking the CK Intron Enhancer Element(VkP-IGKV1-393-Ck-M; VkP-O12-dell)

The construct described in Example 28 was modified by removing the CKintron enhancer element, located in the intergenic region between thehuman J region and the rat CK region by standard PCR modification andDNA cloning methodologies (GeneArt, GmBH). The resulting expressioncassette is shown in FIG. 35 and named VkP-IGKV1-39/J-Ck-Δ1(VkP-O12-dell).

An outline of the pVkP-O12-dell vector and the targeting strategy isdepicted in FIGS. 45B and 46B. The vector was introduced into ES cellsfollowing standard procedures (see, Example 33).

Example 31 Construction of a VK Promoter-Driven Expression ConstructContaining an IGKV1-39 Clone Lacking the CK Intron Enhancer Element anda Truncated 3′ CK Enhancer Element (VkP-IGKV1-39/J-Ck-Δ2; VkP-O12-del2)

The construct described in Example 40 was modified by truncating the 3′CK enhancer element and deleting part of the intergenic region 3′ of therat Ck gene, to remove potential inhibitory elements. This was achievedby removing the intergenic sequence between an EcoRV site (located 3′ ofthe rat Ck gene) and the Ncol site present in the 3′ enhancer (5993 bp)and further removing the sequence between the 3′ enhancer BstXI site andthe BstXI site 3′ of the 3′ enhancer (474 bp) using standard methods.The resulting expression cassette is shown in FIG. 26 and namedVkP-IGKV1-39/J-Ck-Δ2 (VkP-O12-del2).

An outline of the pVkP-O12-del2 vector and the targeting strategy isdepicted in FIGS. 25C and 26C. The vector was introduced into ES cellsfollowing standard procedures (see, Example 33).

Example 32 Expression of VK Constructs in Cell Lines

The constructs described in Examples 28-31 are tested for their abilityto produce light chain proteins in the myeloma cell lines MPC11 (ATCCCCL167), B-cell lymphoma WEHI231 (ATCC CRL-1702), the T-cell lymphomaEL4 (ATCC TIB-39) and in HEK293 (ATCC CRL1573). The enhancer andpromoter elements in the construct enable expression in the B-cell linesbut not in cell lines derived from other tissues. After transfection ofthe cell lines using purified linearized DNA and Fugene6 (Roche) cellsare cultured for transient expression. Cells and supernatant areharvested and subjected to SDS-PAGE analysis followed by westernblotting using a specific anti-rat-C-kappa antibody. Supernatants areanalyzed in ELISA for secreted L chains using the anti-rat CK antibody(Beckton Dickinson #550336).

Example 33 Generation of Transgenic ES Lines

All constructs as described in Examples 22, 23, 24, 25, 28, 29, 30 and31 were used to generate individual stable transgenic ES lines by meansof homologous recombination. The methods for generation of transgenic ESlines via homologous recombination are known in the field (e.g., Egganet al., PNAS 98:6209-6214; J. Seibler, B. Zevnik, B. Kiiter-Luks, S.Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N Faust, G.Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kühn, F. Schwenk(2003), Nucleic Acids Res., Feb 15; 31(4):e12; Hogan et al. (Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y.), pp. 253-289).

For all constructs described in Examples 5 and 6, and Examples 9-12, theRMCE ES cell line (derived from mouse strain129S6B6F1-Gt(ROSA)26Sortm10Arte) was grown on a mitotically inactivatedfeeder layer comprised of mouse embryonic fibroblasts (MEF) in DMEM HighGlucose medium containing 15% FBS (PAN 1302-P220821). LeukemiaInhibitory Factor (Chemicon ESG 1107) was added to the medium at aconcentration of 900 U/mL. For manipulation, 2×10⁵ ES-cells were platedon 3.5 cm dishes in 2 ml medium. Directly before transfection, 2 mlfresh medium was added to the cells. Three μl Fugene6 Reagent (Roche;Catalog No. 1 814 443) was mixed with 100 μ1 serum free medium (OptiMEMI with Glutamax I; Invitrogen; Catalog No. 51985-035) and incubated forfive minutes. One hundred μl of the Fugene/OptiMEM solution was added to2 μg circular vector and 2 μg CAGGS-Flp and incubated for 20 minutes.This transfection complex was added dropwise to the cells and mixed.Fresh medium was added to the cells the following day. From day 2onwards, the medium was replaced daily with medium containing 250 μg/mLG418 (Geneticin; Invitrogen; Catalog No. 10131-019). Seven days aftertransfection, single clones were isolated, expanded, and molecularanalyzed by Southern blotting according to standard procedures.

For each construct, analysis of multiple clones by restriction enzymedigestion of genomic DNA of single clones followed by hybridization with5′ probes, 3′ probes, and internal probes resulted in clones thatcomprised a correct, single insertion at the correct position in theRosa26 locus. An example is provided in FIGS. 39A-39C.

Example 34 Generation of Transgenic Mouse Strains

All ES cell lines that were generated and verified for theirmodifications as described in Example 33 were used to generate stabletransgenic mice by means of tetraploid recombination. The methods areknown in the field. In general, after administration of hormones,superovulated Balb/c females were mated with Balb/c males. Blastocystswere isolated from the uterus at dpc 3.5. For microinjection,blastocysts were placed in a drop of DMEM with 15% FCS under mineraloil. A flat tip, piezo actuated microinjection-pipette with an internaldiameter of 12-15 micrometers was used to inject 10-15 targeted C57BL/6N.tac ES cells into each blastocyst. After recovery, injectedblastocysts were transferred to each uterine horn of 2.5 days postcoitum, pseudopregnant NMRI females. Chimerism was measured in chimeras(GO) by coat color contribution of ES cells to the Balb/c host(black/white). Highly chimeric mice were bred to strain C57BL/6 females.Depending on the project requirements, the C57BL/6 mating partners arenon-mutant (W) or mutant for the presence of a recombinase gene(Flp-Deleter or Cre-deleter or CreER inducible deleter or combination ofFlp-deleter/CreER). Germline transmission was identified by the presenceof black, strain C57BL/6, offspring (G1).

For example, ESC clone IgVK1-39 2683 8 (see, Examples 5 and 14) wasinjected in a total of 62 blastocysts in three independent experiments.Three litters were obtained with a total of six pups. All pups werechimeric. Three heterozygous offspring pups were obtained that were usedfor further crossing.

ESC Clone Kappa 2692 A-C10 (see, Examples 3 and 14) was injected in atotal of 54 blastocysts in three independent experiments. Three litterswere obtained with a total of eleven pups, of which ten were chimeric.Eight heterozygous offspring pups were obtained that were used forfurther crossing.

ESC Clone Kappa 2692 B-C1 (see, Examples 3 and 14) was injected in atotal of 51 blastocysts in three independent experiments. Two litterswere obtained with a total of six pups, of which four were chimeric.Three heterozygous offspring pups were obtained that were used forfurther crossing.

Example 35 Breeding

This example describes the breeding for obtaining mice that containtransgenic expression cassettes as described Example 33 and knock-outmice in which the endogenous lambda and kappa loci have been silenced.The localization of V-lambda on chromosome 16 and CD19 on chromosome 7allow standard breeding procedures. The breeding of the co-localized Vklocus and Rosa26 locus on chromosome 6 with a distance of about 24 cMrequires special attention during the screening as only a percentage ofthe offspring shows crossover in a way that both modifications arebrought together on one chromosome.

All four loci have to be combined in a single mouse strain that is homo-or heterozygous for CD19-cre (not described) and modified Rosa26transgene and homozygous for the other loci. Breeding is performed bystandard breeding and screening techniques as appropriate and offered bycommercial breeding companies (e.g., TaconicArtemis).

Example 36 Immunizations of Mice

Primary and booster immunization of mice are performed using standardprotocols.

To validate the transgenic expression of human rearranged Vκ O12(IGKV1-39)—rat Cκ light chains (see, Examples 5, 14-16) in B cells fromCD19-HuVκ1 mice and to assess its impact on VH repertoire size,diversity of VH family usage and V(D)J recombination after immunization,the CD19-Huκ1 transgenic mice are immunized with tetanus toxin vaccine(TT vaccine) and VH sequence diversity of randomly picked clones fromCD19-HuVκ1 mice are compared with TT-immunized wt mice and CD19-CreHuVk1 negative littermates. Data on the SHM frequency of the human VκO12 transgene in the immunized mice are obtained. A diverse collectionof at least 40 TT-specific, clonally-unrelated mAbs containing the humanVκ O12 are recovered from CD19-HuVκ1 mice by phage display.

For this, three adult CD19-HuVκ1 mice are vaccinated with TT vaccineusing standard immunization procedures. After immunization, serum titersare measured using TT specific ELISA (TT: Statens Serum Institute, Art.no. 2674) and spleen suspensions subjected to cell sorting by the FACSprocedure after staining with a rat Cκ-specific monoclonal antibody toisolate transgenic B cells (clone RG7/9.1; BD Pharmingen# 553901, Lot#06548). RNA from rat Cκ-positive B cells are extracted and the resultingcDNA material used for library building and SHM analysis.

The standard monoclonal mouse anti-rat Cκ antibody (clone RG7/9.1; BDPharmingen# 553901, Lot# 06548) is used in FACS analysis of transgeneexpressing B cells (Meyer et al. (1996), Int. Immunol. 8:1561). Theclone RG7/9.1 antibody reacts with a monotypic (common) kappa chaindeterminant This anti-rat Cκ antibody (clone RG7/9.1 (BD Pharmingen#553901, Lot# 06548) is labeled with R-phycoerythrin (PE) using the LYNXrapid conjugation kit according to the manufacturer's instructions forFACS analysis and sorting. The labeled antibody is firstly tested byflow cytometry for binding to rat Cκ-containing functional light chainproteins produced into transiently transfected HEK-293T cells; theun-conjugated antibody serves as a positive control. Two otherantibodies shown to bind to rat Cκ by ELISA and Western-blot (see,Example 26) are tested as well by flow cytometry.

Fab-phage display library building is carried out with a set ofoptimized degenerate PCR primers designed to amplify C57BL/6 VH genes;the minimal library size is 10⁶ clones, and minimal insert frequency is80%. The vector used, MV1043 (FIGS. 28 and 37A-37Z), contains the humanVκ O12 fused to a human Cκ region. The rat Cκ is therefore exchanged forthe human counterpart in the library generation process.

Before selection, VH sequencing of 96 randomly picked clones isperformed to validate VH repertoire diversity that is compared todiversity obtained from an unselected library previously generated usingthe same procedures from BALB/c mice immunized with TT. A library fromC57B1/6 wt mice that are immunized in the same way allows diversitycomparison between two preselected libraries sharing the same vaccineand the same genetic background.

Several independent selections are performed on TT coated inimmunotubes. Variables that may be included are selections usingbiotinylated antigens in solution or selections on captured TT. Based onthe number and diversity of ELISA-positive clones obtained in the firstselections, decisions on additional rounds of selection are made. Clonesare considered positive when >3× positive over a negative control clone.Positive clones are analyzed by ELISA against a panel of negativecontrol antigens to verify antigen specificity. The aim is to identifyat least 40 unique VH regions, as based on unique CDR3 sequences andV_(H)DJ_(H) rearrangements.

Amplification of the cDNA material from rat Cκ-positive sorted B cellsis performed with a PCR forward primer specific to the human leadersequence and a PCR reverse primer specific to the rat Cκ sequence, in aregion not redundant with the mouse Cκ sequence, as reported in a recentstudy (Brady et al. (2006), JIM 315:61). Primer combinations andannealing temperatures are firstly tested on cDNA from HEK-293T cellstransfected with 0817676_pSELECT_0815426=pSELECT vector with IGKV1-39DNA cassette (see, Example 26).

The amplification products is cloned in pJET-1 vector and after XL1-bluetransformation, 96 colonies are sequenced for assessing VL SHM frequencyby direct comparison to the Vκ O12 (IGKV1-39) germline sequence. The R/Sratio method, as described in the study on human TT-specific antibodies(de Kruif et al. (2009), J. Mol. Biol. 387:548) allows discriminationbetween random mutations and antigen-driven mutations that occurred onVL sequences.

Example 37 Immunofluorescent Analysis of B Cell Populations inTransgenic Mouse Lines

This example describes the use of antibodies and flow cytometry toanalyze B cell populations in primary (bone marrow) and secondary(spleen, peritoneal) lymphoid organs and blood. Methods and reagents aredescribed in Middendorp et al. (2002), J. Immunol. 168:2695; andMiddendorp et al. (2004), J. Immunol. 172:1371. For analysis of early Bcell development in bone marrow, cells were surface stained withcombinations of antibodies (Becton Dickinson) specific for B220, CD19,CD25, IgM, IgD, mouse Ckappa, mouse Clambda and rat Ckappa to detectpro-B cells, pre-B cells, large pre-B cells, early and late immature Bcells and recirculating B cell populations expressing the transgene ontheir surface. DAPI staining (Invitrogen) was included to exclude deadcells from the analysis and FC block (Becton Dickinson) to inhibitantibody interaction with Fc receptors on myeloid cells. For analysis ofsurface transgene expression on B cell populations in peripherallymphoid organs and blood, cells were stained with combinations ofantibodies (Becton Dickinson) specific for B220, CDS, CD19, CD21, CD23,IgM, IgD, mouse Ckappa, mouse Clambda and rat Ckappa. DAPI staining wasincluded to exclude dead cells from the analysis and FC block to inhibitantibody interaction with Fc receptors on myeloid cells. In addition,combinations of antibodies (Becton Dickinson) specific for CD3, CD4,CD11b, CD11c and NK1.1 were included to determine if transgeneexpression occurred in cell types outside of the B cell compartment.

Three mice heterozygous for the human IGKV1-39/rat Ckappa transgene andheterozygous for the CD19-Cre transgene on a C57BL6 background(HuVk1/CD19-Cre) were analyzed. As controls for the FACS analysis, threelittermate mice wild-type for the human IGKV1-39/rat Ckappa transgeneand heterozygous for the CD19-Cre transgene on a C57BL6 background(CD19-Cre) and two C57BL6/NTac mice (Wt) were included. All animals wereallowed to acclimatize in the animal facility for one week beforeanalysis and all mice were male and six weeks of age. Lymphocytes wereisolated from the femurs, spleens, peritoneal cavity and blood of miceusing conventional techniques as previously described (Middendorp et al.(2002), J. Immunol. 168:2695; and Middendorp et al. (2004), J. Immunol.172:1371). Antibodies were pre-combined as shown in Table 10 andstaining was carried out in 96-well plates. Incubation with thePE-conjugated anti-rat C kappa (described above) was carried out beforestaining with the rat anti-murine antibodies to avoid non-specificbinding. After completion of cell staining, labeled cells were analyzedon a Becton Dickinson LSR II FACS machine and the acquired data analyzedwith FLowJo® software (v6.4.7).

Transgenic mice were similar in weight, appearance and activity towild-type mice. No gross anatomical alterations were observed during theharvesting of tissues. No difference was observed in the numbers of Bcells in the bone marrow (BM) and spleen (Table 11) or in the numbers ofB cells, T cells and myeloid cells in peripheral organs betweentransgenic and wild-type mice. In addition, the frequency or proportionof the cells in the different lymphocyte developmental pathways was notaltered in transgenic mice when compared to wild-type mice. Thus in thedouble transgenic (HuVk1/CD19-Cre) and transgenic (CD19-Cre) micelymphoid and most importantly B cell development was indistinguishablefrom wild-type mice.

In the peripheral lymphoid organs, staining with the transgene specificantibody (anti-ratCkappa-PE) was only observed in the B cellpopulations. T cell, myeloid cell and NK cell populations were allnegative for surface expression of the transgene in the spleen (FIGS.48A-48C). In contrast, in cells stained with the pan B cell markers B220and CD19 all cells were shifted to the right in the FACS plot indicatingcell surface expression of the transgene (FIGS. 49A and 49B). A similartransgene-specific staining was measured in CDS⁺B1 cells of theperitoneum, a developmentally distinct population of B cells (FIGS. 50Aand 50B).

Differentiation of B cells from multilineage precursors to mature Bcells occurs in the bone marrow. In the lymphocytes analyzed from thebone marrow, extracellular and transgene expression was not detectablein the earliest B cell progenitors the pro- and pre-B cell consistentwith the pattern of normal light chain expression (FIGS. 51A-1-51B-2).Transgene expression first becomes detectable in immature B cells, thedevelopmental stage at which the germline murine light chain undergoesrearrangement and is expressed at the cell surface in the context of thepreselected heavy chain (FIGS. 51A-1-51B-2). Consistent with thestaining in the spleen transgenic light chain expression is alsodetected on mature recirculating B cells (FIGS. 51A-1-51B-2). Thus theCD19-Cre driven expression of the transgene is consistent with thenormal pattern of light chain expression. The staining with theendogenous light chain- specific antibody is more intense than that ofthe transgene-specific light chain antibody. This may indicate a higherexpression level of the endogenous light chain, a more sensitivestaining with the endogenous light chain-specific antibody or acombination of both. Importantly, the intensity of the surfaceexpression of the transgenic light chain is correlated with bothendogenous light chain and IgM surface expression as observed instaining of circulating B cells in the blood (FIGS. 52A and 52B).

Thus, overall this analysis demonstrates that expression of the humanIGKV1-39/Ckappa transgene is restricted to the B cell compartment andthe temporal regulation of its expression is similar to the endogenouskappa and lambda light chains resulting in normal development of all Bcell populations. The apparent lower level of expression of thetransgene could be explained by the strength of the promoter incomparison to the promoter and enhancers present on endogenous lightchain genes or by a delay in transgene expression that gives theendogenous light chains a competitive advantage in pairing with therearranged heavy chain. This is consistent with the observation that asB cells mature the relative intensity of transgene staining increasescompared to the endogenous light chains. In addition, the observationthat B cells numbers are normal and that every surface Ig+ B cellco-expresses an endogenous and transgenic light chain supports theconclusion that the IGKV1-39 variable region is capable of pairing witha normal repertoire of different murine heavy chain variable regions.Concluded from this analysis was that insertion of the IGKV1-39/ratCkappa transgene driven by the CD19-Cre activated CAGGS promoter in theRosa locus facilitates timely and B cell-specific expression of thetransgene and that the transgene is capable of pairing with a normalrepertoire of murine heavy chains.

Example 38 EPIBASE® T-Cell Epitope Profile for IGKV1-39

The protein sequence of IGKV1-39 (FIGS. 37A-37Z, human germlineIGKV1-39/J Protein) was scanned for the presence of putative HLA classII restricted epitopes, also known as T_(H)-epitopes. For this,Algonomics' EPIBASE® platform was applied to IGKV1-39. In short, theplatform analyzes the HLA binding specificities of all possible 10-merpeptides derived from a target sequence (Desmet et al. (1992), Nature356:539-542; Desmet et al. (1997), FASEB J. 11:164-172; Desmet et al.(2002), Proteins 48:31-43; Desmet et al. (2005), Proteins 58:53-69).Profiling is done at the allotype level for 20 DRB1, 7 DRB3/4/5, 13 DQand 7 DP, i.e., 47 HLA class II receptors in total (see, Table 5).EPIBASE® calculates a quantitative estimate of the free energy ofbinding ΔG_(bind) of a peptide for each of the 47 HLA class IIreceptors. These data were then further processed as follows:

Free energies were converted into Kd-values through ΔG_(bind)=RT ln(Kd).

Peptides were classified as strong (S), medium (M), weak and non (N)binders. The following cutoffs were applied:

S: strong binder: Kd<0.1 μM.

M: medium binder: 0.1 μM≦Kd<0.8 μM.

N: weak and non-binder: 0.8 μM≦Kd.

Peptides corresponding to self-peptides were treated separately. Thelist of self-peptides was taken from 293 antibody germline sequences.They are referred to as “germline-filtered” peptides.

S- and M-peptides are mapped onto the target sequence in so-calledepitope maps; S-affinities are plotted quantitatively; M-values arepresented qualitatively. As a general overview of the results, Table 6lists the number of strong and medium binders in the analyzed proteins,for the groups of HLA class II receptors corresponding to the DRB1, DQ,DP and DRB3/4/5 genes. Counting was done separately for strong andmedium affinity binders. Peptides binding to multiple allotypes of thesame group were counted as one. Values between brackets refer togermline-filtered peptides. In Table 7, the sequence is shown in aformat suitable for experimental work. The sequence is broken down inconsecutive 15-mers overlapping by 12 residues. For each 15-mer, thepromiscuity is listed (the number of allotypes out of a total of 47 forwhich the 15-mer contains a critical binder), as well as the impliedserotypes. The EPIBASE® profile and epitope maps are shown in FIGS.41A-41C and 42.

It was concluded that IGKV1-39 contains no strong non-self DRB1 binders.Typically, significantly more binders were found for DRB1 than for otherHLA genes. This is in agreement with experimental evidence thatallotypes belonging to the DRB1 group are more potent peptide binders.Medium strength epitopes for DRB1 allotypes are expected to contributeto the population response, and cannot be disregarded. Again, nonon-self DRB1 binders were found in IGKV1-39.

In the humoral response raised against an antigen, the observed T_(H)cell activation/proliferation is generally interpreted in terms of theDRB1 specificity. However, one cannot ignore the possible contributionof the DRB3/4/5, DQ and DP genes. Given the lower expression levels ofthese genes as compared to DRB1, the focus was on the class of strongepitopes for DRB3/4/5, DQ and DP. “Critical epitopes” are those epitopesthat are strong binders for any DRB1, DRB3/4/5, DQ or DP allotype or aremedium binders for DRB1. IGKV1-39 contains no strong or medium non-selfbinders for DRB3/4/5, DQ, or DP.

A number of peptides are also present in germline sequences (valuesbetween brackets in Table 6). Such peptides may very well bind to HLAbut they are assumed to be self and, hence, non-immunogenic. In total,six strong and 16 medium germline-filtered DRB1 binders were found inIGKV1-39. Framework region 1 up to framework region 3 is an exact matchfor germline V-segment VKI 2-1-(1) O12 (VBase), a.k.a. IGKV1-39*01(IMGT). Framework region 4 is an exact match for germline J-segment JK1(V-base) a.k.a. IGKJ1*01(IMGT). It is hardly surprising that thesesegments do not contain any non-self epitopes.

Example 39 Production Characteristics of IGKV1-39

There is a great demand for antibody discovery platforms that yieldtherapeutic antibodies that are thermodynamically stable and give goodexpression yields. These characteristics are important in ensuring thestability of the drug substance during production and after injection ofthe drug product into the patient. In addition good expression yieldsimpact directly on the cost of drug manufacture and thus pricing,patient access and profitability. Virtually all therapeutic antibodiesin clinical use today are composed of human IgG1 and kappa constantregions but use different heavy and light chain variable regions thatconfer specificity. Human variable heavy and light chain domains can bedivided into families that have greater than 80% sequence divergence.When rearranged examples of these families in germline configuration arecombined and compared for stability and yield it is clear that the genefamilies are not equal in terms of biophysical properties. In particularV_(H)3, V_(H)1 and V_(H)5 have favourable stability for the heavy chainsand Vk1 and Vk3 have the best stability and yield of light chains Inaddition when mutations are introduced as part of the somatichypermutation process they can interfere with V_(H)/V_(L) pairing. Toassess the effect that different light chain genes with different ratesof mutation have on the production characteristics of a fixed V_(H)chain, a Fab phage display library was built of light chains (kappa andlambda) from six naive healthy donors combined with a panel of 44 TTbinding heavy chains from immunized donors. After one round of selectionTT binding Fab clones were isolated. Several of these shared the sameV_(H) gene as the TT clone PG1433 in combination with different lightchains. The Fab light chain fragments were recloned into a kappaexpression vector and transfected in combination with DNA encoding theheavy chain of PG1433 into 293 cells and specific IgG productionmeasured by ELISA. As demonstrated in Table 8 the selected clonescontaining PG1433 V_(H) combined with different light chains had betweenfive- and ten-fold lower protein expression PG1433 V_(H) combined withIGKV1-39. Note that all of the light chains contained amino acidmutations within their coding regions that might disrupt V_(H) paringand reduce production stability. Thus, in addition to reducing thechances of unwanted immunogenicity, it is expected that the use of thelight chain IGKV1-39 without mutations contributes to improvedproduction stability and yields of various specificity-contributingV_(H) genes. Indeed stable clones generated by the transfection ofdifferent V_(H) genes all paired with IGKV1-39 are able to be passagedextensively and still retain robust production characteristics as shownin Table 9.

Example 40 Generation of Mice Expressing Fully Human VH and VL Regions

Transgenic mice described herein are crossed with mice that alreadycontain a human VH locus. Examples of appropriate mice comprising ahuman VH locus are disclosed in Taylor et al. (1992), Nucleic Acids Res.20:6287-95; Lonberg et al. (1994), Nature 368:856-9; Green et al.(1994), Nat. Genet. 7:13-21; Dechiara et al. (2009), Methods Mol. Biol.530:311-24.).

After crossing and selecting for mice that are at least heterozygous forthe IGKV1-39 transgene and the human VH locus, selected mice areimmunized with a target. VH genes are harvested as describedhereinabove. This method has the advantage that the VH genes are alreadyfully human and thus do not require humanization.

Example 41 Isolation, Characterization, OLIGOCLONICS® Formatting andProduction of Antibodies Targeting Human IL6 for Treatment of ChronicInflammatory Diseases such as Rheumatoid Arthritis

A spleen VH repertoire from transgenic mice that are immunized withhuman recombinant IL6 is cloned in a phage display Fab vector with asingle human IGKV1-39-C kappa light chain (identical to the mousetransgene) and subjected to panning against the immunogen human IL6.Clones that are obtained after two to four rounds of panning areanalyzed for their binding specificity. VH genes encoding IL6-specificFab fragments are subjected to sequence analysis to identify uniqueclones and assign VH, DH and JH utilization. The Fab fragments arereformatted as IgG1 molecules and transiently expressed. Unique clonesare then grouped based on non-competition in binding assays andsubjected to affinity and functional analysis. The most potent anti-IL6IgG1 mAbs are subsequently expressed as combinations of two, three, fouror five heavy chains comprising different VH-regions in theOLIGOCLONICS® format, together with one IGKV1-39-C-based kappa lightchain and tested in vitro for complex formation with IL-6. TheOLIGOCLONICS® are also tested in vivo for clearance of human IL-6 frommice. An OLIGOCLONICS® with the most potent clearance activity is chosenand the murine VH genes humanized according to conventional methods. Thehumanized IgG1 are transfected into a mammalian cell line to generate astable clone. An optimal subclone is selected for the generation of amaster cell bank and the generation of clinical trial material.

Many of the protocols described here are standard protocols for theconstruction of phage display libraries and the panning of phages forbinding to an antigen of interest and are described, for example, inAntibody Phage Display: Methods and Protocols (2002), Editor(s) PhilippaM. O'Brien, Robert Aitken, Humana Press, Totowa, N.J., US.

Immunizations: Transgenic mice receive three immunizations with humanIL6 every two weeks using the adjuvant Sigma titerMax according tomanufacturer's instructions.

RNA isolation and cDNA synthesis: Three days after the lastimmunization, spleens and lymphnodes from the mice are removed andpassed through a 70 micron filter into a tube containing PBS pH 7.4 togenerate a single cell suspension. After washing and pelleting oflymphocytes, cells are suspended in TRIzol LS Reagent (Invitrogen) forthe isolation of total RNA according to the manufacturer's protocol andsubjected to reverse transcription reaction using 1 microgram of RNA,Superscript III RT in combination with dT20 according to manufacturer'sprocedures (Invitrogen).

The generation of Fab phage display libraries is carried out asdescribed in Example 21.

Selection of phages on coated immunotubes: Human recombinant IL6 isdissolved in PBS in a concentration of 5 μg/ml and coated to MaxiSorpNunc-Immuno Tube (Nunc 444474) overnight at 4° C. After discarding thecoating solution, the tubes are blocked with 2% skim milk (ELK) in PBS(blocking buffer) for one hour at Room Temperature (RT). In parallel,0.5 ml of the phage library is mixed with 1 ml blocking buffer andincubated for 20 minutes at room temperature. After blocking the phages,the phage solution is added to the IL6-coated tubes and incubated fortwo hours at RT on a slowly rotating platform to allow binding. Next,the tubes are washed ten times with PBS/0.05% TWEEN®-20 followed byphage elution by incubating with 1 ml 50 mM glycine-HCl pH 2.2 tenminutes at RT on rotating wheel and directly followed by neutralizationof the harvested eluent with 0.5 ml 1 M Tris-HCl pH 7.5.

Harvesting phage clones: A 5 ml XL1-Blue MRF (Stratagene) culture atO.D. 0.4 is added to the harvested phage solution and incubated for 30minutes at 37° C. without shaking to allow infection of the phages.Bacteria are plated on Carbenicillin/Tetracycline 4% glucose 2*TY platesand grown overnight at 37° C.

Phage production: Phages are grown and processed as described by Krameret al. 2003 (Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) usingVCSM13 as helper phage strain.

Phage ELISA: ELISA plates are coated with 100 microliters humanrecombinant IL6 per well at a concentration of 2.5 micrograms/ml in PBSovernight at 4° C. Plates coated with 100 microliters thyroglobulin at aconcentration of 2 micrograms/ml in PBS are used as a negative control.Wells are emptied, dried by tapping on a paper towel, filled completelywith PBS-4% skimmed milk (ELK) and incubated for one hour at roomtemperature to block the wells. After discarding the block solution,phage minipreps pre-mixed with 50 μl blocking solution are added andincubated for one hour at RT. Unbound phages are subsequently removed byfive washing steps with PBS-0.05% TWEEN®-20. Bound phages are detectedby incubating the wells with 100 microliters anti-M13-HRP antibodyconjugate (diluted 1/5000 in blocking buffer) for one hour at roomtemperature. Free antibody is removed by repeating the washing steps asdescribed above, followed by TMB substrate incubation until colordevelopment was visible. The reaction is stopped by adding 100microliters of 2 M H2SO4 per well and analyzed on an ELISA reader at 450nm emission wavelength.

Sequencing: Clones that give signals at least three times above thebackground signal are propagated, used for DNA miniprep procedures (see,procedures Qiagen miniPrep manual) and subjected to nucleotide sequenceanalysis. Sequencing is performed according to the Big Dye 1.1 kitaccompanying manual (Applied Biosystems) using a reverse primer(CH1_Rev1, Table 1) recognizing a 5′ sequence of the CH1 region of thehuman IgG1 heavy chain (present in the Fab display vector MV1043, FIGS.28 and 37A-37Z). The sequences of the murine VH regions are analyzed fordiversity of DH and JH gene segments.

Construction and expression of chimeric IgG1: Vector MV1057 (FIGS.37A-37Z and 47) was generated by cloning the transgene (IGKV1-39) Lchain fragment into a derivative of vector pcDNA3000Neo (Crucell,Leiden, The Netherlands) that contains the human IgG1- and kappaconstant regions. VH regions are cloned into MV1057 and nucleotidesequences for all constructs are verified according to standardtechniques. The resulting constructs are transiently expressed inHEK293T cells and supernatants containing chimeric IgG1 are obtained andpurified using standard procedures as described before (M. Throsby 2006,J. Virol. 80:6982-92).

IgG1 binding and competition analysis: IgG1 antibodies are titrated inELISA using IL6-coated plates as described above and an anti-human IgGperoxidase conjugate. Competition ELISAs to group antibodies based onepitope recognition are performed by incubating Fab phages together withIgG1 or with commercial antibodies against IL6 (e.g., Abcam cat. no.ab9324) in IL6-coated plates, followed by detection of bound Fab phageusing an anti-M13 peroxidase conjugate.

IgG1 affinity measurements: The affinities of the antibodies to IL6 aredetermined with the Quantitative kinetic protocol on the Octet(ForteBio). Antibodies are captured onto an Anti-Human IgG Fc Capturebiosensor and exposed to free IL6 and analyzed using proprietarysoftware to calculate the Kd of each antibody.

Functional activity of IL6 antibodies: To test the ability of theselected antibodies to inhibit binding between IL6 and IL6 receptor(IL6R), an ELISA based assay is used. Various concentrations of antibodyare mixed with a fixed concentration (10 ng/ml) of biotinylated IL6 asdescribed by Naoko et al. 2007, Can. Res. 67:817-875. The IL6-antibodyimmune complex is added to immobilized IL6R. The binding of biotinylatedIL6 to IL6R is detected with horseradish peroxidase-conjugatedstreptavidin. The reduction of ELISA signal is a measurement ofinhibition. As positive control for inhibition of binding between IL6and IL6R either anti-IL6R antibody (Abcam cat. no. ab34351; clone B-R6)or anti IL6 antibody (Abcam cat. no. ab9324) is used.

In vitro blocking activity of the selected anti-IL6 antibodies ismeasured in a proliferation assay using the IL6-dependent cell line7TD1. Briefly, cells are incubated with different concentrations ofhuman IL6 with or without the anti-IL6 antibody. The available amount ofIL6 determines the degree of proliferation. Thus if an added antibodyblocks IL6 binding the proliferation readout is reduced compared to anon binding antibody control. Proliferation is measured by theincorporation of 5-bromo-2′-deoxy-uridine (BrdU) into the DNA using theBrdU proliferation kit (Roche cat. no. 11444611001) according to themanufacturer's instructions.

Generation of anti-IL6 OLIGOCLONICS®: The most potent anti-IL6antibodies are selected from each epitope group. The expressionconstructs expressing these antibodies are transfected into HEK293Tcells in non-competing groups of three in different ratios (1:1:1;3:1:1; 1:3:1; 1:1:3; 3:3:1; 1:3:3; 3:1:3; 10:1:1; 1:10:1; 1:1:10;10:10:1; 1:10:10; 10:1:10; 3:10:1; 10:3:1; 1:10:3; 3:1:10; 10:1:3;1:3:10). Antibody containing supernatants are harvested and purified andcharacterized as above.

Complex formation and in vivo clearance of anti-IL6 OLIGOCLONICS®: Tomeasure the ability of anti-IL6 OLIGOCLONICS® to form immune complexesand to analyze these complexes Size Exclusion Chromatography (SEC) isused according to the approach disclosed by Min-Soo Kim et al. (2007),JMB 374:1374-1388, to characterize the immune-complexes formed withdifferent antibodies to TNFα. Different molar ratios of the anti-IL6OLIGOCLONICS® are mixed with human IL6 and incubated for 20 hours at 4°C. or 25° C. The mixture is analyzed on an HPLC system fitted with asize exclusion column; different elution times are correlated tomolecular weight using a molecular weight standards.

The ability of antibodies to form complexes with IL6 is correlated withtheir ability to rapidly clear the cytokine from the circulation invivo. This is confirmed by measuring the clearance of radiolabelled IL6from mice. Briefly, female, six- to eight-week-old Balb/c mice areobtained and 18 hours before the experiment, the animals are injectedintravenously (IV) via the lateral tail vein with different doses ofpurified anti-IL6 OLIGOCLONICS®. On day 0, the mice are injected IV with50 microliters of radiolabeled IL-6 (1×10E7 cpm/mL) under the sameconditions. Blood samples (approximately 50 microliters) are collectedat several time intervals and stored at 4° C. The samples arecentrifuged for five minutes at 4000×g and the radioactivity of theserum determined. All pharmacokinetic experiments are performedsimultaneously with three animals for each treatment.

Generation of anti-IL6 OLIGOCLONICS® stable clones and preclinicaldevelopment: A lead anti-IL6 OLIGOCLONICS® is selected based on the invitro and in vivo potency as determined above. The murine VH genes arehumanized according to standard methods and combined with the fullyhuman IGKV1-39 light chain in an expression vector as described above.Examples of humanization methods include those based on paradigms suchas resurfacing (E. A. Padlan et al. (1991), Mol. Immunol. 28:489),superhumanization (P. Tan, D. A., et al. (2002), J. Immunol. 169:1119)and human string content optimization (G. A. Lazar et al. (2007), Mol.Immunol. 44:1986). The three constructs are transfected into PER.C6®cells at the predetermined optimal ratio (described above) under theselective pressure of G418 according to standard methods. A stable highproducing anti-IL6 OLIGOCLONICS® clone is selected and a working andqualified master cell bank generated.

TABLE 1 List of primers DO- Primer Sequence 0012 CH_Rev1TGCCAGGGGGAAGACCGATG (SEQ ID NO: 4) 0656 MVH-1GCCGGCCATGGCCGAGGTRMAGCTTCAGGAGTCAGGAC (SEQ ID NO: 5) 0657 MVH-2GCCGGCCATGGCCGAGGTSCAGCTKCAGCAGTCAGGAC (SEQ ID NO: 6) 0658 MVH-3GCCGGCCATGGCCCAGGTGCAGCTGAAGSASTCAGG (SEQ ID NO: 7) 0659 MVH-4GCCGGCCATGGCCGAGGTGCAGCTTCAGGAGTCSGGAC (SEQ ID NO: 8) 0660 MVH-5GCCGGCCATGGCCGARGTCCAGCTGCAACAGTCYGGAC (SEQ ID NO: 9) 0661 MVH-6GCCGGCCATGGCCCAGGTCCAGCTKCAGCAATCTGG (SEQ ID NO: 10) 0662 MVH-7GCCGGCCATGGCCCAGSTBCAGCTGCAGCAGTCTGG (SEQ ID NO: 11) 0663 MVH-8GCCGGCCATGGCCCAGGTYCAGCTGCAGCAGTCTGGRC (SEQ ID NO: 12) 0664 MVH-9GCCGGCCATGGCCCAGGTYCAGCTYCAGCAGTCTGG (SEQ ID NO: 13) 0665 MVH-10GCCGGCCATGGCCGAGGTCCARCTGCAACAATCTGGACC (SEQ ID NO: 14) 0666 MVH-11GCCGGCCATGGCCCAGGTCCACGTGAAGCAGTCTGGG (SEQ ID NO: 15) 0667 MVH-12GCCGGCCATGGCCGAGGTGAASSTGGTGGAATCTG (SEQ ID NO: 16) 0668 MVH-13GCCGGCCATGGCCGAVGTGAAGYTGGTGGAGTCTG (SEQ ID NO: 17) 0669 MVH-14GCCGGCCATGGCCGAGGTGCAGSKGGTGGAGTCTGGGG (SEQ ID NO: 18) 0670 MVH-15GCCGGCCATGGCCGAKGTGCAMCTGGTGGAGTCTGGG (SEQ ID NO: 19) 0671 MVH-16GCCGGCCATGGCCGAGGTGAAGCTGATGGARTCTGG (SEQ ID NO: 20) 0672 MVH-17GCCGGCCATGGCCGAGGTGCARCTTGTTGAGTCTGGTG (SEQ ID NO: 21) 0673 MVH-18GCCGGCCATGGCCGARGTRAAGCTTCTCGAGTCTGGA (SEQ ID NO: 22) 0674 MVH-19GCCGGCCATGGCCGAAGTGAARSTTGAGGAGTCTGG (SEQ ID NO: 23) 0675 MVH-20GCCGGCCATGGCCGAAGTGATGCTGGTGGAGTCTGGG (SEQ ID NO: 24) 0676 MVH-21GCCGGCCATGGCCCAGGTTACTCTRAAAGWGTSTGGCC (SEQ ID NO: 25) 0677 MVH-22GCCGGCCATGGCCCAGGTCCAACTVCAGCARCCTGG (SEQ ID NO:2 6) 0678 MVH-23GCCGGCCATGGCCCAGGTYCARCTGCAGCAGTCTG (SEQ ID NO: 27) 0679 MVH-24GCCGGCCATGGCCGATGTGAACTTGGAAGTGTCTGG (SEQ ID NO: 28) 0680 MVH-25GCCGGCCATGGCCGAGGTGAAGGTCATCGAGTCTGG (SEQ ID NO: 29) 0681 ExtMVH-1CAGTCACAGATCCTCGCGAATTGGCCCA

ATGGCCSANG (SEQ ID NO: 30) 0682 ExtMVH-2 CAGTCACAGATCCTCGCGAATTGGCCCA

ATGGCCSANC (SEQ ID NO: 31) 0683 MJH-Rev! GGGGGTGTCGTTTTGGCTGAGGAGAC 

 GTGG (SEQ ID NO: 32) 0684 MJH-Rev2 GGGGGTGTCGTTTTGGCTGAGGAGAC 

 GTGG (SEQ ID NO: 33) 0685 MJH-Rev3 GGGGGTGTCGTTTTGGCTGCAGAGAC 

 AGAG (SEQ ID NO: 34) 0686 MJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC 

 GAGG (SEQ ID NO: 35) 0687 ExtMJH-Rev1& GGGGGTGTCGTTTTGGCTGAGGAGAC 

 GTGG (SEQ ID NO: 36) 0688 ExtMJH-Rev2in GGGGGTGTCGTTTTGGCTGAGGAGAC 

 GTGG (SEQ ID NO: 37) 0690 ExtMJH-Rev3 GGGGGTGTCGTTTTGGCTGAGGAGAC 

 AGAG (SEQ ID NO: 38) 0691 ExtMJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC 

 GAGG (SEQ ID NO: 39)

TABLE 2 Phage ELISA signal levels as measured at 450 nm. TT-coatedplates represent plates that were coated with tetanus toxoid.Thyroglobulin-coated plates are used as negative controls. 10/10 and15/15 indicate the number of wash steps with PBS-Tween ® during panningprocedures. The 10/10 tetanus toxoid and 10/10 thyroglobulin plates andthe 15/15 tetanus toxoid and 15/15 thyroglobulin plates are duplicatesfrom each other except for the coating agent. OD values higher thanthree times the background are assumed specific. 1 2 3 4 5 6 7 8 9 10 1112 TT-coated plate 10/10 washings A 0.139 0.093 0.089 0.121 0.117 0.5980.146 0.115 0.18 0.155 0.543 0.601 B 0.136 0.404 0.159 0.187 0.489 0.1340.216 0.092 0.222 0.108 0.181 0.484 C 0.197 0.526 0.09 0.213 0.395 0.1550.108 0.12 0.183 0.136 0.092 0.866 D 0.143 0.258 0.101 0.422 0.088 0.2430.485 0.251 0.304 0.198 0.478 0.091 E 0.445 0.169 0.526 0.481 0.2060.285 0.111 0.119 0.128 0.2 0.118 0.098 F 0.237 0.291 0.594 0.139 0.2060.565 0.543 0.091 0.136 0.227 0.228 0.099 G 0.459 0.102 0.152 0.6590.203 0.452 0.152 0.133 0.094 0.102 0.375 0.098 H 0.341 0.623 0.7450.415 0.682 0.527 0.655 0.114 0.258 0.284 0.685 0.113 TT-coated plate15/15 washings A 0.247 0.582 0.421 0.428 0.133 0.082 0.262 0.079 0.3430.414 0.095 0.292 B 0.065 0.364 0.073 0.042 0.049 0.071 0.046 0.1030.078 0.057 0.048 0.155 C 0.081 0.044 0.066 0.082 0.225 0.444 0.2030.362 0.122 0.047 0.052 0.309 D 0.092 0.11 0.59 0.22 0.33 0.544 0.0580.159 0.047 0.174 0.086 0.05 E 0.469 0.577 0.206 0.304 0.13 0.749 0.4310.062 0.167 0.049 0.056 0.049 F 0.846 0.07 0.561 0.656 0.882 0.094 0.3830.13 0.152 0.098 0.134 0.048 G 0.537 0.052 0.49 0.105 0.337 0.193 0.5140.294 0.068 0.35 0.525 0.05 H 0.061 0.306 0.157 0.853 0.054 0.534 0.1020.235 0.441 0.412 0.565 0.061 Thyroglobulin-coated plate 10/10 washingsA 0.047 0.051 0.045 0.043 0.051 0.044 0.046 0.042 0.047 0.048 0.049 0.05B 0.042 0.042 0.042 0.042 0.043 0.041 0.041 0.042 0.043 0.045 0.0420.046 C 0.044 0.043 0.043 0.044 0.043 0.044 0.043 0.042 0.043 0.0410.044 0.046 D 0.045 0.044 0.044 0.044 0.045 0.046 0.045 0.056 0.0450.049 0.048 0.73 E 0.046 0.045 0.046 0.044 0.045 0.044 0.044 0.044 0.0470.046 0.047 0.926 F 0.048 0.045 0.044 0.046 0.044 0.043 0.044 0.0460.046 0.046 0.046 0.792 G 0.051 0.048 0.045 0.045 0.044 0.043 0.0480.045 0.048 0.051 0.045 0.053 H 0.064 0.05 0.049 0.047 0.05 0.051 0.0470.046 0.047 0.047 0.047 0.056 Thyroglobulin-coated plate 15/15 washingsA 0.036 0.049 0.045 0.044 0.046 0.047 0.046 0.042 0.042 0.043 0.0420.041 B 0.045 0.042 0.041 0.043 0.043 0.043 0.045 0.045 0.047 0.0480.044 0.045 C 0.049 0.047 0.047 0.046 0.046 0.046 0.045 0.047 0.0460.045 0.045 0.052 D 0.047 0.049 0.048 0.048 0.048 0.048 0.047 0.0520.048 0.046 0.048 0.456 E 0.049 0.047 0.047 0.047 0.047 0.049 0.0470.048 0.047 0.046 0.048 0.412 F 0.05 0.047 0.046 0.046 0.046 0.046 0.0460.046 0.046 0.047 0.048 0.528 G 0.05 0.048 0.045 0.045 0.046 0.049 0.0480.046 0.053 0.049 0.05 0.057 H 0.057 0.05 0.046 0.045 0.047 0.049 0.0470.047 0.046 0.047 0.053 0.048

TABLE 3Protein sequence analysis of ELISA positive tetanus toxoid binders. CDR3 sequence,CDR3 length, VH family members and specific name, JH origin and DH origin of theclones is indicated. CDR3  V Gene CDR3/SEQ ID NO: length VH DH JH familyHGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAYYTYDEKAWFAY (SEQ ID NO: 40) 15 musIGHV192 DSP2.11 JH3 mouse VH7183HGAFYTYDEKPWFAY (SEQ ID NO: 41) 15 musIGHV192 IGHD2-14*01 JH3 mouseVH7183 HISYYRYDEEVSFAY (SEQ ID NO: 42) 15 musIGHV192 IGHD2-14*01JH3 mouse VH7183 HISYYRYDEEVSFAY (SEQ ID NO: 42) 15 musIGHV192IGHD2-14*01 JH3 mouse VH7183 GWRAFAY (SEQ ID NO: 43)  7 musIGHV131DSP2.9 JH3 mouse VH7183 GWRAFAY (SEQ ID NO: 43)  7 musIGHV131 DSP2.9JH3 mouse VH7183 GWRAFAY (SEQ ID NO: 43)  7 musIGHV131 DSP2.9 JH3 mouseVH7183 DRGNYYGMDY (SEQ ID NO: 44) 10 musIGHV178 DSP2.1 JH4 mouse VH7183LGDYYVDWFFAV (SEQ ID NO: 45) 12 musIGHV165 DFL16.1 JH1 mouse VH7183NFPAWFAF (SEQ ID NO: 46)  8 musIGHV547 DST4.3inv JH3 mouse VJH558NFPAWFAY (SEQ ID NO: 46)  8 musIGHV547 DSP2.1 JH3 mouse VJH558NFPAWFVY (SEQ ID NO: 46)  8 musIGHV547 DSP2.1 JH3 mouse VJH558SFTPVPFYYGYDWYFDV (SEQ ID NO: 47) 17 musIGHV532 DSP2.3 JH1 mouse VJH558SFTPVPFYYGYDWYFDV (SEQ ID NO: 47) 17 musIGHV532 DSP2.3 JH1 mouse VJH558SDYDWYFDV (SEQ ID NO: 48)  9 musIGHV286 DSP2.2 JH1 mouse VJH558SDYDWYFDV (SEQ ID NO: 48)  9 musIGHV286 DSP2.2 JH1 mouse VJH558DSKWAYYFDY (SEQ ID NO: 49) 10 musIGHV532 DST4.3 JH2 mouse VJH558GDYTGYGMDY (SEQ ID NO: 50) 10 musIGHV125 DSP2.13 JH4 mouse VHSM7GDYTGYGMDY (SEQ ID NO: 50) 10 musIGHV125 DSP2.13 JH4 mouse VHSM7GGYDGYWFPY (SEQ ID NO: 51) 10 musIGHV125 DSP2.9 JH3 mouse VHSM7

TABLE 4 Vector combinations that were transfected to HEK293T. CombinedConc. Code HC vector LC vector vector Prep name (μg/ml) A x0817676_pSELECT_0815426 x PIGKV1-39/ — (IGKV1-39) P1 B x0817678_pSELECT_0815427 x PIGLV2-14/ — (IGLV2-14) P1 C MV11100817676_pSELECT_0815426 x PMV1110/ 11.0 (IGKV1-39) IGKV1-39/P1 D MV11100817678_pSELECT_0815427 x PMV1110/ 15.4 (IGLV2-14) IGLV2-14/P1 E x xMG1494 MG1494/P2 16.1

TABLE 5 HLA allotypes considered in T_(H)-epitope profiling. Thecorresponding serotypes are shown, as well as allotype frequencies inthe Caucasian population (Klitz et al. (2003), Tissue Antigens 62:296-307; Gjertson and Terasake (eds) in: HLA 1997; Gjertson and Terasake(eds) in: HLA 1998; Castelli et al. (2002), J. Immunol. 169: 6928-6934).Frequencies can add up to more than 100% since each individual has twoalleles for each gene. If all allele frequencies of a single gene wereknown, they would add up to slightly less than 200% due to homozygousindividuals. HLA type Serotype Population % DRB1*0101 DR1 17.4 DRB1*0102DR1 4.9 DRB1*0301 DR17(3) 21.2 DRB1*0401 DR4 11.5 DRB1*0402 DR4 3.1DRB1*0404 DR4 5.5 DRB1*0405 DR4 2.2 DRB1*0407 DR4 <2 DRB1*0701 DR7 23.4DRB1*0801 DR8 3.3 DRB1*0802 DR8 <2 DRB1*0901 DR9 <2 DRB1*1101 DR11(5) 17DRB1*1104 DR11(5) 5.7 DRB1*1201 DR12(5) 3.1 DRB1*1301 DR13(6) 15.4DRB1*1302 DR13(6) 10.8 DRB1*1401 DR14(6) 4.2 DRB1*1501 DR15(2) 13.2DRB1*1601 DR16(2) 5.5 DRB3*0101 DR52 24.6 DRB3*0202 DR52 43 DRB3*0301DR52 10 DRB4*0101 DR53 25.5 DRB4*0103 DR53 21 DRB5*0101 DR51 15.8DRB5*0202 DR51 5.7 DQA1*0101/DQB1*0501 DQ5(1) 20.5 DQA1*0102/DQB1*0502DQ5(1) 2.6 DQA1*0102/DQB1*0602 DQ6(1) 26.5 DQA1*0102/DQB1*0604 DQ6(1)6.7 DQA1*0103/DQB1*0603 DQ6(1) 11 DQA1*0104/DQB1*0503 DQ5(1) 4DQA1*0201/DQB1*0202 DQ2 20.9 DQA1*0201/DQB1*0303 DQ9(3) 7.2DQA1*0301/DQB1*0301 DQ7(3) 12.5 DQA1*0301/DQB1*0302 DQ8(3) 18.3DQA1*0401/DQB1*0402 DQ4 4.5 DQA1*0501/DQB1*0201 DQ2 24.6DQA1*0501/DQB1*0301 DQ7(3) 20.9 DPA1*0103/DPB1*0201 DPw2 19.9DPA1*0103/DPB1*0401 DPw4 65.1 DPA1*0103/DPB1*0402 DPw4 24.3DPA1*0201/DPB1*0101 DPw1 6.3 DPA1*0201/DPB1*0301 DPw3 <2DPA1*0201/DPB1*0501 DPw5 <2 DPA1*0201/DPB1*0901 — 2.4

TABLE 6 T_(H) epitope counts for IGKV1-39. Peptides binding to multipleHLAs of the same group (DRB1, DRB3/4/5, DP, DQ) are counted as one.Values between brackets refer to germline-filtered peptides. DRB1DRB3/4/5 DQ DP Strong Medium Strong Medium Strong Medium Strong MediumMerus IGKV1-39 0 (+6) 0 (+16) 0 (+0) 0 (+5) 0 (+3) 0 (+9) 0 (+0) 0 (+9)

TABLE 7 Mapping of EPIBASE ®predictions for Mercus IGKVI-39 in the classical 15-mer peptideformat. This table shows the allotype count of critical epitopes (SEQ ID NOs: 52-83) andimplicated serotypes for each of the 15-mers spanning the Merus IGKVI-39 sequenceStart Allotype 15mer position 15-mer sequence count Implicated serotypes 1  1 DIQMTQSPSSLSASV  6 DR1, DR4, DR7, DR9  2  4 MTQSPSSLSASVGDR  5DR1, DR4, DR9  3  7 SPSSLSASVGDRVTI  0  4 10 SLSASBGDRYTITCR  0  5 13ASVGDRVTITCRASQ  0  6 16 GDRVTITCRASQSIS  2 DR11(5), DR7  7 19VTITCRASQSISSYL  4 DQ2, DR11(5), SR4, DR7  8 22 TCRASQSISSYLNWY  2DQ2, DR4  9 25 ASQSISSYLNWYQQK  5 DR13(6), DR15(2), DR4 10 28SISSYLNWYQQKPGK  8 DR12(5), DR13(6), DR15(2), DR16(2), DR4, DR8 11 31SYLNWYQQKPGKAPK 10 DR1, DR12(5), DR16(2), DR4, DR51, DR8 12 34NWYQQKPGKAPKLLI  9 DR1, DR15(2), DR4, DR51, DR8 13 37 QQKPGKAPKLLIYAA  7DQ4, DR1, DR11(5), DR15(2), DR51, DR8 14 40 PGKAPKLLIYAASSL  7DQ4, DR1, DR11(5), DR4, DR8 15 43 APKLLIYAASSLQSG 15DR1, DR11(5), DR12(5), DR13(6), DR14(6),  DR15(2), DR4, DR51, DR8, DR916 46 LLIYAASSLQSGVPS 15 DR1, DR11(5), DR12(5), DR13(6), DR14(6), DR15(2), DR4, DR51, DR8, DR9 17 49 YAASSLQSGVPSRFS  1 DR15(2) 18 52SSLQSGVPSRFSGSG  1 DR15(2) 19 55 QSGVPSRFSGSGSGT  0 20 58VPSRFSGSGSGTDFT  0 21 61 RFSGSGSGTDFTLTI  0 22 64 GSGSGTDFTLTISSL  1DR52 23 67 SGTDFTLTISSLQPE  4 DR4, DR52, DR7, DR9 24 70 DFTLTISSLQPEDFA 4 DQ2, DR4, DR7, DR9 25 73 LTISSLQPEDFATYY  1 DQ2 26 76 SSLQPEDFATYYCQQ 0 27 79 QPEDFATYYCQQSYS  1 DR4 28 82 DFATYYCQQSYSTPP  5 DR4, DR51, DR729 85 TYYCQQSYSTPPTFG  4 DR4, DR51, DR7 30 88 CQQSYSTPPTFGQGT  0 31 91SYSTFFTFGQGTKVE  0 32 94 TPPTFGQGTKVEIK  0

TABLE 8 The V_(H) gene from PG1433 paired with various light chain geneswith differing rates of amino acid mutation were compared for productionlevels with the original clone containing the IGKV1-39 gene. Number ofLight chain amino acid concentration IgG name gene mutations (μg/ml)PG1433 1-39 0 63, 45.5, 38.6 (avg = 49) PG1631 1-12 4 10.5 PG1632 1-27 79.3 PG1634 1D-12 10 10.8 PG1635 1D-33 6 10.2 PG1642 1-5 8 7.1 PG1644 1-93 7.8 PG1650 1D-39 3 9.1 PG1652 2D-28 3 7.1 PG1653 3-15 14 7 PG1654 3-202 5.2 PG1674 1-40 7 8.2 PG1678 2-11 2 8.1 PG1680 2-14 15 10.8 PG1682 3-113 9.9 PG1683 6-57 6 13.9

TABLE 9 Parameters of stability for stable clones containing thegermline IGKV1-39 gene. Culture Batch Maximum IVC at days at Avg pdt instarted at viable cell maximum IgG start previous population density(×10⁶ concentration Subclone batch run 14 days ±SD % avg doublingscells/ml) % avg (10⁹ cells/hr/L) % avg B38.1 21 35 5.1 99 15 3 91 530 9240 41 1.3 115 31 3.7 112 568 99 79 36 0.2 101 62 3.2 97 627 109 avg 363.3 575 B38.4 21 35 1 101 15 2.2 114 424 134 40 35 0.3 101 29 1.9 98 24778 79 34 0.2 99 59 1.7 88 278 88 avg 35 1.9 316 B38.15 21 35 1.6 106 162.5 90 497 101 40 32 0.3 97 30 3.7 134 557 114 79 31 0.2 94 63 2.1 76415 85 avg 33 2.8 490 B38.30 21 38 9.2 97 15 1.6 81 335 89 40 51 2.7 13130 2.7 137 472 125 79 40 0.7 103 64 1.6 81 325 86 avg 39 2.0 377 B224.1823 34 2.6 100 17 3.1 103 507 103 42 37 0.7 109 33 3.6 120 575 117 81 340.2 100 63 2.3 77 393 80 avg 34 3.0 492 B224.47 23 32 0.4 102 17 3.5 98695 109 42 33 0.3 105 31 3.6 101 578 91 81 31 0.2 98 64 3.5 101 634 100avg 32 3.6 636 B224.53 23 33 0.5 100 17 3.9 110 553 99 42 32 0.4 97 333.7 105 605 108 81 33 0.1 100 63 3 85 525 94 avg 33 3.5 561 B224.59 2336 0.6 104 16 4.3 115 750 107 42 34 0.2 99 30 4.4 118 779 111 81 33 0.396 61 2.5 67 583 83 avg 35 3.7 704 B280.3 23 34 0.8 105 17 4.3 105 840108 42 32 0.4 98 33 4 98 841 108 81 31 0.1 95 67 4 98 660 85 avg 33 4.1780 B280.12 23 36 1.7 104 15 2 72 426 77 42 37 0.7 107 30 3.2 116 673122 81 33 0.2 96 64 3.1 112 552 100 avg 35 2.8 550 B280.21 23 32 0.6 10218 3.1 103 550 97 42 31 0.4 98 34 3.4 113 589 104 81 31 0.4 98 66 2.5 83566 100 avg 32 3.0 568 B280.36 23 33 1 99 17 3 81 596 75 42 36 0.5 10730 4.6 124 1168 146 81 34 0.3 101 62 3.5 95 635 79 avg 34 3.7 800Culture days at Maximum IgG start qAb concentration Correlationcorrelation correlation Subclone batch run (pg/cell/day) % avg (mg/L) %avg TF FH TH B38.1 21 9.5 97 122 79 0.99 0.95 0.92 40 10.5 107 188 122 10.99 0.99 79 9.4 96 154 100 0.97 0.99 0.96 avg 9.8 155 B38.4 21 14.2 116141 127 1 0.96 0.97 40 12.5 102 96 86 1 1 1 79 9.9 81 97 87 0.99 1 0.99avg 12.2 111 B38.15 21 7.9 99 97 93 0.99 0.95 0.93 40 7.3 91 114 109 10.97 0.97 79 8.8 110 102 98 0.96 0.96 0.99 avg 8 104 B38.30 21 14.5 112100 71 0.99 1 0.99 40 13.9 107 206 147 1 0.99 0.99 79 10.6 82 114 810.98 0.98 0.99 avg 13 140 B224.18 23 15.8 98 208 81 1 0.99 0.99 42 18.1112 318 124 1 0.94 0.95 81 14.6 90 244 95 1 1 0.99 avg 16.2 257 B224.4723 22.5 114 387 122 0.99 0.93 0.89 42 20 101 357 112 0.99 0.92 0.95 8116.8 85 209 66 1 0.99 0.99 avg 19.8 318 B224.53 23 20.6 102 372 114 0.980.82 0.85 42 24.3 121 379 116 0.98 0.88 0.94 81 15.4 77 231 71 0.99 0.890.94 avg 20.1 327 B224.59 23 16.4 106 301 104 0.99 0.78 0.84 42 14.6 95344 119 0.98 0.92 0.96 81 15.2 99 224 77 0.97 0.99 0.96 avg 15.4 290B280.3 23 13 109 293 117 0.99 0.98 0.95 42 12.3 103 292 116 0.99 0.980.98 81 10.5 88 169 67 0.99 0.98 1 avg 11.9 251 B280.12 23 5.8 95 64 810.98 0.98 0.98 42 6.2 101 96 121 1 0.97 0.97 81 6.4 104 78 98 0.98 0.980.98 avg 6.1 79 B280.21 23 9.1 128 112 93 0.97 0.92 0.93 42 3.6 51 137113 1 0.98 0.99 81 8.6 121 114 94 0.97 0.99 1 avg 7.1 121 B280.36 23 10186 143 156 1 0.99 0.98 42 5.6 104 124 135 1 0.98 0.97 81 0.56 10 8 90.97 0.98 1 avg 5.4 92

TABLE 10 Antibody mixtures used for staining of lymphocyte populations.Stainings Mixtures Facs Work 1st 2nd 3rd Final # tubes # Monoclonaldilution volume step step step diltion A Spleen 1 1-8 CD21^(FITC) 640320 0.50 Ckappa rat^(PE) 160 2.00 CD19^(PerCP-Cy5.5) 640 0.50CD23^(PE-Cy7) 50 1:20 6.40 1000 DAPI Ckappa mouse^(BIO-APC) 100 1:503.20 APC 5000 Clambda mouse^(BIO-APC) 100 1:30 3.20 APC 3000B220^(Alex-700) 160 2.00 FC block 400 0.80 Spleen 2  9-16 IgD^(FITC) 640640 1.00 BM 17-24 Ckappa rat^(PE) 160 4.00 CD19^(PerCP-Cy5.5) 500 1.28IgM^(PE-Cy7) 640 1.00 DAPI Ckappa mouse^(BIO-APC) 100 1:50 6.40 APC 5000Clambda mouse^(BIO-APC) 100 1:30 6.40 APC 3000 B220^(Alex-700) 160 4.00FC block 400 1.60 Spleen 3 25-32 Ckappa mouse^(FITC) 400 320 0.80 Ckapparat^(PE) 160 2.00 CD19^(PerCP-Cy5.5) 500 0.64 IgM^(PE-Cy7) 640 0.50 DAPIClambda mouse^(BIO-APC) 100 1:30 3.20 APC 3000 B220^(Alex-700) 160 2.00FC block 400 0.80 Spleen 4 33-40 Ckappa mouse^(FITC) 400 640 1.60 41-48lambda^(FITC) 600 1.07 PP Ckappa rat^(PE) 160 4.00 CD19^(PerCP-Cy5.5)500 1.28 IgM^(PE-Cy7) 640 1.00 DAPI IgD^(A647) 1280 0.50 B220^(Alex-700)160 4.00 PNA^(BIO-SAV-APC-Cy7) 300 2.13 APC-Cy7 FC block 400 1.60 PC 549-56 IgM^(FITC) 160 320 2.00 Ckappa rat^(PE) 160 2.00CD19^(PerCP-Cy5.5) 500 0.64 Ckappa mouse^(BIO-PE-Cy7) 100 1:50 3.20PE-Cy7 5000 Clambda mouse^(BIO-PE-Cy7) 100 1:30 3.20 PE-Cy7 3000 DAPICD5^(APC) 320 1.00 B200^(Alex-700) 160 2.00 FC block 400 0.80 BM 6 57-64IgM^(FITC) 160 640 4.00 Ckappa rat^(PE) 160 4.00 CD19^(PerCP-Cy5.5) 5001.28 Ckappa mouse^(BIO-PE-Cy7) 100 1:50 6.40 PE-Cy7 5000 Clambdamouse^(BIO-PE-Cy7) 100 1:30 6.40 PE-Cy7 3000 DAPI CD25^(APC) 80 8.00B220^(Alex-700) 160 4.00 FC block 400 1.60 RAT spleen 7 144 Ckapparat^(PE) 160 80 0.5 rat B220^(FITC) 160 0.5 Spleen 8  97-104 cytCD3^(FITC) 320 320 1 cyt Ckappa rat^(PE) 80 4.00 cyt CD11c^(PE-TexasRED)75 4.27 cyt NK1.1^(BIO-PE-Cy7) 200 1.6 PE-Cy7 cyt CD19^(PerCP-Cy5.5) 3201 cyt CD4^(APC) 500 0.64 cyt CD11b^(Alex-700) 50 6.40 BM = bone marrow,PC = peritoneal cavity, PP = Peyer's patches.

TABLE 11 Numbers of lymphocytes harvested from the bone marrow andspleen of wild-type and transgenic mice *10e6/ml Total vol Total cellscells (ml) *10⁶ Bone Marrow Wt 18.82 5.05 95.0 Wt 19.24 4.96 95.4CD19-Cre 23.42 5.08 119.0 CD19-Cre 20.58 4.82 99.2 CD19-Cre 25.77 5.15132.7 CD19-Cre/HuVk1 17.71 5.06 89.6 CD19-Cre/HuVk1 12.60 5.33 67.2CD19-Cre/HuVk1 18.13 5.27 95.5 Spleen Wt 41.70 5.36 223.5 Wt 37.85 4.71178.3 CD19-Cre 60.19 3.77 226.9 CD19-Cre 35.06 3.66 128.3 CD19-Cre 80.694.60 371.2 CD19-Cre/HuVk1 51.67 4.48 231.5 CD19-Cre/HuVk1 58.80 6.24366.9 CD19-Cre/HuVk1 24.37 6.25 152.3

What is claimed is:
 1. A method for selecting a single recombinant cellthat expresses a heterogeneous combination of monospecific andbispecific antibodies, or antibody fragments thereof, wherein themonospecific and bispecific antibodies, or antibody fragments thereof,are human, humanized, or deimmunized, and wherein the heterogeneouscombination has specific affinity for two target epitopes, the methodcomprising: carrying out a process for producing the heterogeneouscombination; the process comprising providing three different variableregions consisting of two heavy chain regions and one light chainvariable region in recombinant cells, wherein antigen binding parts ofthe variable regions originate from a single species and wherein onevariable region is able to functionally pair with more than one othervariable region, and under conditions allowing for pairing of variableregions and secretion of the paired regions from the recombinant cellsresulting in the production of said heterogeneous combination, providingtwo target epitopes, and selecting a single recombinant cell from therecombinant cells that produces a heterogeneous combination that bindsthe two target epitopes, wherein the one light chain variable regioncomprises a rearranged human immunoglobulin light chain variable regionconsisting essentially of a germline V and a germline J and encoded by ahuman immunoglobulin light chain V gene segment joined to a humanimmunoglobulin light chain J gene segment.
 2. The method according toclaim 1, wherein said two target epitopes are associated with a diseaseand/or disorder.
 3. The method according to claim 2, further comprising:subjecting a heterogeneous combination to a biological assay indicativeof an effect of the combination on the disease and/or disorder.
 4. Themethod according to claim 1, wherein the one variable region able tofunctionally pair with more than one other variable region does notsignificantly contribute to the resulting binding specificity of theresulting paired regions.
 5. The method according to claim 1, whereineach variable region can only pair with one other variable region. 6.The method according to claim 1, wherein two of the three variableregions are part of one single chain Fv.
 7. The method according toclaim 1, wherein the expression of two of the variable regions is underthe direction of different control elements.
 8. The method according toclaim 7, wherein the different control elements lead to differentialexpression.
 9. The method according to claim 8, wherein the differentialexpression is different in levels of expression and/or time ofexpression.
 10. The method according to claim 1, wherein the combinationcomprises two monospecific antibodies produced in the single recombinantcell.
 11. The method according to claim 1, further comprising: producingan expression system comprising polynucleotides encoding variableregions, said producing comprising: synthesizing polynucleotidesencoding variable regions, expressing said polynucleotides and allowingthe expression products to pair, and selecting polynucleotides encodingvariable regions having desired pairing behavior, so as to producepolynucleotides encoding variable regions.
 12. The method according toclaim 1, further comprising: expressing the heterogeneous combination ofmonospecific and bispecific antibodies, or antibody fragments thereof,resulting in the production of said heterogeneous combination in thesingle recombinant cell.
 13. The method according to claim 1, whereinthe human immunoglobulin light chain V gene segment joined to the humanimmunoglobulin light chain J gene segment is obtained from a transgenicmouse.
 14. The method according to claim 13, wherein the three differentvariable regions consisting of two heavy chain regions and one lightchain variable region are encoded by a transgenic mouse.
 15. The methodaccording to claim 14, wherein the genome of the transgenic mousecomprises a transgene comprising the human immunoglobulin light chain Vgene segment joined to the human immunoglobulin light chain J genesegment, wherein the joined human V/J gene segments encode therearranged human immunoglobulin light chain variable region, and whereinthe transgenic mouse, in response to the two target epitopes, producesantibodies with immunoglobulin light chains comprising the rearrangedhuman light chain variable region and a murine light chain constantregion, paired with a diversity of immunoglobulin heavy chains whichbind the two target epitopes.
 16. The method according to claim 1,wherein a first heavy chain of the two heavy chain regions is obtainedby immunizing a first mouse with a first target epitope of the twotarget epitopes and wherein a second heavy chain of the two heavy chainregions is obtained by immunizing a second mouse with a second targetepitope of the two target epitopes.
 17. The method according to claim 1,wherein the three different variable regions consisting of two heavychain regions and one light chain variable region are encoded by atransgenic mouse.
 18. The method according to claim 17, wherein thegenome of the transgenic mouse comprises a transgene comprising thehuman immunoglobulin light chain V gene segment joined to the humanimmunoglobulin light chain J gene segment, wherein the joined human V/Jgene segments encode the rearranged human immunoglobulin light chainvariable region, and wherein the transgenic mouse, in response to thetwo target epitopes, produces antibodies with immunoglobulin lightchains comprising the rearranged human light chain variable region and amurine light chain constant region, paired with a diversity ofimmunoglobulin heavy chains which bind the two target epitopes.
 19. Amethod for selecting a single recombinant cell that expresses aheterogeneous combination of monospecific and bispecific antibodies, orantibody fragments thereof, wherein the monospecific and bispecificantibodies, or antibody fragments thereof, are human, humanized, ordeimmunized, and wherein the heterogeneous combination has specificaffinity for two target epitopes, the method comprising: producing a setof antibody combinations from a set of recombinant cells by a processcomprising: pairing, in the set of recombinant cells, three differentvariable regions consisting of two heavy chain variable regions, and onelight chain variable region comprising a rearranged human immunoglobulinlight chain variable region encoded by a human immunoglobulin lightchain V gene segment joined to a human immunoglobulin light chain J genesegment, wherein antigen binding parts of the variable regions originatefrom a single species and wherein one variable region is able tofunctionally pair with more than one other variable region, andsecreting the paired regions from the recombinant cells to produce anantibody combination, testing the set antibody combinations to identitya single heterogeneous combination that has specific affinity for thetwo target epitopes, and isolating from the set of recombinant cells asingle recombinant cell that produces the single heterogeneouscombination.